Polypeptides having L-asparaginase activity

ABSTRACT

Disclosed are polypeptides which originate from mammal, having L-asparaginase activity. The polypeptides are easily prepared by applying recombinant DNA techniques to DNAs encoding the polypeptides and they exert satisfactory effects in the treatment and/or the prevention for diseases caused by tumor cells dependent on L-asparagine, and cause no substantial serious side effects even when administered to humans in relatively-high dose.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No. 08/869,927, filed Jun. 5, 1997, the entire contents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to L-asparagine amidohydrolytic enzymes, more particularly, to polypeptides which originate from mammal, having L-asparaginase activity.

2. Description of the Prior Art

L-Asparaginase (EC 3.5.1.1) is an enzyme which catalyzes the hydrolytic reaction of L-asparagine into L-aspartic acid and ammonia. The studies on the antitumor activity of L-asparaginase started from the following reports: J. G. Kidd et al. described the inhibitory action of guinea pig sera on cells of lymphomas in "The Journal of Experimental Medicine", Vol.98, pp.565-582 (1953), and J. D. Broome et al. evidenced in "Nature", Vol.191, pp.1,114-1,115 (1961), that the L-asparaginase activity of the guinea pig sera was responsible for the inhibitory action. It is now understood that the inhibitory action is caused by the lack of L-asparagine, an essential nutrient to proliferate and survive for some tumor cells which defect L-asparagine synthetase activity, such as acute lymphocytic leukemia, but not for normal cells. The hydrolysis of L-asparagine by L-asparaginase in patients with such tumor cells induces selective death of the tumor cells, resulting in the treatment of malignant tumors.

L-Asparaginase has been studied energetically for its actual use as an antitumor agent, and one derived from Escherichia coli is now in use as a therapeutic agent for leukemia and lymphoma. However, L-asparaginase from Escherichia coli is merely an external protein for human, and repetitive administration of conventional compositions with such L-asparaginase may cause serious side effects such as anaphylaxis shock, urticaria, edema, wheeze and dyspnea. These compositions are inevitably restricted with respect to administration dose and frequency. Therefore, some proposals to reduce or even diminish such side effects have been given.

As a first proposal, Japanese Patent Kokai No.119,082/79 discloses a chemically modified L-asparaginase from Escherichia coli, in which at least 65% amino acids are blocked with 2-O-substituted polyethylene glycol-4,6-dichloro-S-triazine. As a second proposal, human L-asparaginases are disclosed in Japanese Patent Kokai Nos.320,684/92 and 19,018/80, where the L-asparaginases are respectively obtained from cultures of human cell lines and human urine. While the first proposal has an advantage of that the L-asparaginase from Escherichia coli is easily obtainable on an industrial scale, it has a disadvantage of that the modifying reaction is difficult to control and the side effects couldn't be eliminated completely. While the second proposal has an advantage of that unlike L-asparaginase from Escherichia coli, the L-asparaginases from human may not substantially induce antibodies even when administered to patients, it has a disadvantage of that it is not easy to obtain the L-asparaginases in a desired amount by the processes disclosed in Japanese Patent Kokai Nos.320,684/92 and 19,018/80.

Recently, recombinant DNA technology has advanced remarkably. If a DNA which encodes a desired polypeptide is once isolated, it is relatively easy to obtain a transformant which produces the polypeptide by constructing a recombinant DNA, comprising the DNA and a self-replicable vector, followed by introducing the recombinant DNA into a host, such as a microorganism, animal- or plant-cell. The polypeptide is obtainable in a desired amount from the culture of the transformant. However, no DNA which encodes mammalian L-asparaginase was isolated, and no mammalian L-asparaginase was produced by recombinant DNA techniques.

Therefore, it has been in great demand to isolate DNAs which encode active L-asparaginases originating from mammal and establish processes to prepare the L-asparaginases on a large-scale by applying the recombinant DNA techniques to the isolated DNAs.

SUMMARY OF THE INVENTION

In view of foregoing, the first object of the present invention is to provide a polypeptide which originates from mamma, having L-asparaginase activity.

The second object of the present invention is to provide a DNA which encodes the polypeptide.

The third object of the present invention is to provide a recombinant DNA which containing a DNA which encodes the polypeptide and a self-replicable vector.

The fourth object of the present invention is to provide a transformant obtainable by introducing a DNA which encodes the polypeptide into a host.

The fifth object of the present invention is to provide a process to prepare the polypeptide by using the transformant.

The sixth object of the present invention is to provide an agent for susceptive diseases, containing the polypeptide as an effective ingredient.

The first object of the present invention is attained by polypeptides which originate from mammal, having L-asparaginase activity.

The second object of the present invention is attained by DNAs which encode the polypeptides.

The third object of the present invention is attained by recombinant DNAs containing DNA which encode the polypeptides and a self-replicable vector.

The fourth object of the present invention is attained by transformants obtainable by introducing the DNAs into appropriate hosts.

The fifth object of the present invention is attained by a process to prepare the polypeptides which comprises culturing the transformants and collecting the produced polypeptides from the resultant cultures.

The sixth object of the present invention is attained by agents for susceptive diseases, containing the polypeptides as effective ingredients.

BRIEF EXPLANATION OF THE ACCOMPANYING DRAWINGS

FIG. 1 is a scheme of the over lap extension method.

FIG. 2 is a restriction map of the present recombinant DNA pKGPA/WT.

FIG. 3 is a scheme of the preparation of the present recombinant DNA pBIgGPA/WT. the sense and anti-sense primers used in the PCR reaction are designated as SEQ ID NO:37 and SEQ ID NO:38, respectively. The linkers shown in FIG. 3 are SEQ ID NOs: 39-42.

FIG. 4 is a restriction map of the present recombinant DNA pBIgGPA/WT.

FIG. 5 is a restriction map of the present recombinant DNA pKGPA/D364stp.

FIG. 6 is a restriction map of the present recombinant DNA pKHA/MUT1.

FIG. 7 is a restriction map of the present recombinant DNA pKHA/MUT2.

FIG. 8 is a restriction map of the present recombinant DNA pKHA/MUT3.

FIG. 9 is a restriction map of the present recombinant DNA pKHA/MUT3.

FIG. 9 is a restriction map of the present recombinant DNA pKHA/MUT5.

FIG. 10 is a restriction map of the present recombinant DNA pBIgGPA/D364stp.

FIG. 11 is a restriction map of the present recombinant DNA pBIgHA/MUT1.

FIG. 12 is a restriction map of the present recombinant DNA pBIgHA/MUT2.

FIG. 13 is a restriction map of the present recombinant DNA pBIgHA/MUT3.

FIG. 14 is a restriction map of the present recombinant DNA pBIgHA/MUT4.

Explanation of the symbols are as follows:

The symbols, "Eco RI", "Hin dIII", "Not I" and "Xho I", indicate cleavage sites by restriction enzymes, Eco RI, Hin dIII, Not I and Xho I, respectively.

The symbols, "D364stp", "HA/MUT1", "HA/MUT2", "HA/MUT3" and "HA/MUT5", indicate DNAs encoding the present polypeptides.

The symbol "Ptac" indicates a Tac promotor.

The symbol "rrnBT1T2" indicates a region for transcriptional termination, derived from a ribosomal RNA operon.

The symbol "AmpR" indicates an ampicillin resistant gene.

The symbol "pBR322ori" indicates a replication origin in Escherichia coli.

The symbol "Ig sec" indicates a DNA encoding a polypeptide with a signal sequence for secretion of immunoglobulin.

The symbol "Emsv" indicates an enhancer from long terminal repeats of Moloney Mouse Sarcoma Virus.

The symbol "Pmti" indicates a promotor for Mouse metallothionein I gene.

The symbol "Poly (A)" indicates a polyadenylation signal derived from SV40 virus.

The symbol "BPVI" indicates a genome of a bovine papillomavirus.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors isolated mammalian DNAs encoding L-asparaginases firstly in the world, from guinea pig and human, and succeeded in elucidating their nucleotide sequences. The nucleotide sequences of the DNAs from a guinea pig and human are in SEQ ID NOs:15 and 16, respectively. This information is disclosed in Japanese Patent Application No.42,564/95 (Japanese Patent Kokai No.214,885/96) by the same applicant of this application. The present invention has been made based on the above information, and provides the polypeptides which originate from mammal, having L-asparaginase activity.

The polypeptides of the present invention are not restricted to their sources or origins so far as they originate from mammal and have an L-asparaginase activity. The polypeptides are usually obtainable by the expression of genes originating from mammal, and usually contain amino acid sequences of SEQ ID NOs:1 to 3, wherein the symbol "Xaa" in SEQ ID NO:3 means "glutamine" or "arginine". For example, the polypeptides have any one of amino acid sequences of SEQ ID NOs:4 to 9. In view of the technical level in this field, one or more amino acid residues in SEQ ID NOs:4 to 9 can be substituted relatively easily by different ones without substantial defects of the activity. Despite derived from the same DNA, a variety of polypeptides with an L-asparaginase activity may be obtained as a result of modifications by endogenous enzymes of the hosts after the DNA expression or modifications during purification of the polypeptides, depending on the types of vectors and hosts used to obtain transformants or culturing conditions of the transformants, such as ingredients, compositions, temperatures or pHs. The wording "a variety of polypeptides" includes the polypeptides with deletions and/or additions of one or more amino acids at the N- and/or C-termini thereof, or with glycosylations. In view of these, the present polypeptides include not only the polypeptide with any amino acid sequence of SEQ ID NOs:4 to 9 but also their homologues so long as they have an L-asparaginase activity. The present polypeptides express the activity when exist in multiple forms, preferably, tetramers.

The polypeptides of the present invention can be usually prepared by the recombinant DNA techniques. In general, the polypeptides are obtainable by culturing transformants containing DNAs encoding the polypeptides and collecting the produced polypeptides from the resultant cultures. The transformants are obtainable by introducing such recombinant DNAs as contain any one of the nucleotide sequences of SEQ ID Nos:10 to 15 and a self-replicable vector into appropriate hosts. One or more nucleotides in SEQ ID NOs:10 to 15 can be substituted by different nucleotides without substantial changes of the encoding amino acid sequences with respect to degeneracy of genetic code. To facilitate the expression of the DNA in the hosts, one or more nucleotides in nucleotide sequences which encode the polypeptides or their homologues can be appropriately substituted by different ones. Furthermore, nucleotide sequences which encode and/or don't encode one or more amino acids can be added to the 5'- and/or 3'-termini of the nucleotide sequences.

The DNAs encoding the polypeptides of this invention include those from natural sources and those by synthesized artificially so far as the polypeptides expressed by them have an L-asparaginase activity. The DNAs can be wild-type ones, containing the same nucleotide sequences as those from natural sources, and can be their homologues.

Examples of the wild-type DNAs include DNAs containing the nucleotide sequences of SEQ ID NOs:15. The wild-type DNA is obtainable from natural sources such as guinea pig livers, as disclosed in Japanese Patent Application No.42,564/95 (Japanese Patent Kokai No.214,885/96) by the same applicant of this invention: (a) constructing a cDNA library by applying usual methods to purified poly (A)⁺ RNAs from a guinea pig or human liver as materials, (b) applying the plaque hybridization method to the cDNA library using oligonucleotides as probes synthesized chemically based on partial amino acid sequences of L-asparaginase purified from a guinea pig serum, (c) collecting phage clones containing the DNAs encoding the polypeptides of this invention, and (d) manipulating the collected phage clones in a conventional manner. The wild-type DNA can be synthesized chemically based on SEQ ID NO:15.

Examples of DNA homologues to the wild-type ones include DNAs containing any nucleotide sequence of SEQ ID NOs:10 to 14. DNA homologues containing the nucleotide sequence of SEQ ID NO:10 are obtainable by applying conventional methods in this field, such as PCR method and methods for site-directed mutagenesis, to the wild-type DNA of SEQ ID NO:15 concerning the desired sequence. DNA homologues containing any nucleotide sequence of SEQ ID NOs:11 to 14 are obtainable by the methods such as follows: Firstly, A wild-type DNA with the nucleotide sequence of SEQ ID NO:16 is obtained by the methods as disclosed in Japanese Patent Application No.42,564/95 (Japanese Patent Kokai No.214,885/96) by the same applicant of this invention, i.e., screening a human liver cDNA library. Subsequently, the wild-type DNA is subjected to conventional methods as mentioned above concerning desired sequences to obtain the DNA homologues. The DNA homologues can be synthesized chemically based on the nucleotide sequences of SEQ ID NOs:10 to 14.

The present DNAs can be generally introduced into hosts as in forms of recombinant DNAs. In general, each recombinant DNA comprises one of the present DNAs and a self-replicable vector. The recombinant DNAs can be easily prepared by general recombinant DNA techniques when the DNAs are available. Examples of such self-replicable vectors include pKK223-3, pGEX-2T, pRL-λ, pBTrp2 DNA, pUB110, YEp13, Ti plasmid, Ri plasmid, pBI121, pCDM8, pBPV and BCMGSneo. Among these vectors, pKK223-3, pGEX-2T, pRL-λ, pBTrp2 DNA pUB110 are suitably used to express the present DNAs in prokaryotic cells such as Escherichia coli and Bacillus sp., while YEp13, Ti plasmid, Ri plasmid, pBI121, pCDM8, pBPV and BCMGSneo are suitably used to express the present DNAs in eukaryotic cells such as yeasts and animal- and plant-cells.

To insert the present DNAs into the vectors, conventional methods in this field can be arbitrarily used. Examples of such methods contain the steps of (a) cleaving self-replicable vectors with restriction enzymes, (b) introducing the same cleavage sites, by the same restriction enzymes as used to cleave the vectors, to the 5'- and 3'-termini of the present DNAs by applying polymerase chain reaction to form double-stranded DNAs, (c) cleaving the double-stranded DNAs by the restriction enzymes, and (d) ligating the cleaved vectors with cleaved DNAs by the action of DNA ligases. The recombinant DNAs thus obtained can be easily introduced into appropriate hosts, resulting in limitless replication of the DNAs by culturing the transformants.

The recombinant DNAs according to the present invention can be introduced into appropriate hosts such as Escherichia coli, Bacillus sp., actinomycetes, yeasts and plant-and animal-cells. To introduce the DNAs into Escherichia coli, it can be cultured in the presence of the recombinant DNAs and calcium ion. To introduce them into Bacillus sp., competent cell methods or protoplast methods can be used. To introduce them into animal-cells, DEAE-dextran methods or electroporation methods can be used. Desired transformants can be cloned by applying hybridization methods or by selecting L-asparaginase producing cells from the cultures.

The transformants thus obtained produce the present polypeptides intracellularly or extracellularly when cultured in nutrient culture media. Examples of such media are usually liquid nutrient culture media which generally contain carbon sources, nitrogen sources and minerals, and further contain micronutrients such as amino acids and/or vitamins on demand. The carbon sources usable in the present invention include saccharides such as starch, starch hydrolysates, glucose, fructose and sucrose. The nitrogen sources usable in the present invention include organic and inorganic compounds containing nitrogen, such as ammonia and their salts, urea, nitrates, peptone, yeast extract, defatted soy bean, corn steep liquor and beef extract. Cultures containing the present polypeptides can be obtained by inoculating the transformants into the above media, culturing them at temperatures of 25-65° C. at pHs of 5-8 for about 1-10 days under aerobic conditions by aeration-agitation method, etc.

The cultures can be used intact as agents for susceptive diseases. However, the cultures are usually treated with ultrasonication or cell wall lytic enzymes to disrupt cells, and the present polypeptides are separated by using techniques such as filtration and centrifugation from the cell-disruptants and purified. Alternatively, the polypeptides can be purified from the culture supernatants obtained by removing cells from the cultures by filtration or centrifugation, etc. The present polypeptides can be purified by applying techniques generally used in this field for protein purifications, such as salting out, dialysis, filtration, concentration, gel filtration chromatography, ion-exchange chromatography, affinity chromatography, hydrophobic chromatography, isoelectric focusing and gel electrophoresis, and if necessary, two or more of them can be applied combination to the supernatants which are separated from insoluble substances of cell-disruptants, or to the culture supernatants. The resultant purified solutions polypeptides can be concentrated and/or lyophilized into liquids or solids depending on their final uses.

The following experiments explain the present invention in more detail, and the techniques used therein are conventional ones in this field: For example, the techniques are disclosed by J. Sambrook et al. in "Molecular Cloning, A Laboratory Manual", 2nd edition (1989), published by Cold Spring Harbor Laboratory Press, New York, U.S.A., and by Masami MATSUMURA in "Laboratory Manual for Genetic Engineering" (1988), published by Maruzen Co., Ltd., Tokyo, Japan.

EXPERIMENT 1

Expression of Wild-type DNA

EXPERIMENT 1-1

Expression of Guinea Pig Wild-type DNA

EXPERIMENT 1-1(a)

Preparation of Guinea Pig Wild-type DNA

A guinea pig wild-type DNA encoding L-asparaginase was prepared by the method disclosed in Japanese Patent Kokai No.214,885/96 by the same applicant of this invention. The DNA had the nucleotide sequence of SEQ ID NO:15. A DNA having a polypeptide-encoding region in SEQ ID NO:15, i.e., a sequence of containing the nucleotides 20-1,714 in SEQ ID NO:15, is called "GPA/WT DNA" hereinafter, and the expression product thereof with the amino acid sequence of SEQ ID NO:49 is called "guinea pig wild-type L-asparaginase". SEQ ID NO:17 shows in parallel the nucleotide sequence of GPA/WT DNA and the amino acid sequence (SEQ ID NO:49) encoded thereby.

EXPERIMENT 1-1(b)

Preparation of Recombinant DNA

Ten μl of 10×PCR buffer, one μl of 25 mM dNTP mix, one ng of the human wild-type DNA, obtained in Experiment 1-1(a), as a template were placed in 0.5 ml reaction tube. The mixture was mixed with, as a sense- and anti-sense-primers, an adequate amount of an oligonucleotide chemically synthesized based on the amino acid sequences near the N- and C-termini of SEQ ID NO:15, volumed up with sterilized distilled water to give a total volume of 99.5 μl , and mixed with 0.5 μl of 2.5 units/μl of AmpliTaq DNA polymerase. The nucleotide sequence of the sense primer was 5'-AATCTCGAGCCACCATGGCGCGCGCATCA-3' (SEQ ID NO:19), a nucleotide sequence obtained by adding a common nucleotide sequence in animal cells, as shown by M. Kozak in "Nucleic Acid Research", Vol.15, pp.8,125-8,148 (1987), to the upstream of a region which encodes the N-terminal amino acid sequence of SEQ ID NO:15 and then adding to the further upstream a cleavage site by a restriction enzyme, Xho I. The nucleotide sequence of the anti-sense primer was 5'-CTGCGGCCGCTTATCAGATGGCAGGCGGCAC-3' (SEQ ID NO:20) as a complement to a nucleotide sequence obtained by adding two termination codons to the downstream of a region which encodes the C-terminus of the amino acid sequence of SEQ ID NO:15 and adding a cleavage site by a restriction enzyme, Not I, to the further downstream. The resulting mixture was successively incubated at 94° C. for one min, at 55° C. for one min, and at 72° C. for 3 min, and the series of incubation was repeated 40-times for PCR to amplify DNA. Thus, a DNA containing GPA/WT DNA was obtained and then cleaved by restriction enzymes of Xho I and Not I to obtain an about 1.7 kbp DNA fragment. Twenty-five ng of the DNA fragment was weighed and mixed with 10 ng of a plasmid vector, "pCDM8", commercialized by Invitrogen Corporation, San Diego, U.S.A., which had been cleaved by restriction enzymes of Xho I and Not I. To the DNA mixture thus obtained was added an equal volume of the solution I in "LIGATION KIT VERSION 2" commercialized by Takara Shuzo, Tokyo, Japan, and incubated at 16° C. for 2 hours to obtain a replicable recombinant DNA, "pCGPA/WT".

The recombinant DNA pCGPA/WT was introduced into an Escherichia coli MC1061/P3 strain, commercialized by Invitrogen Corporation, San Diego, U.S.A., by competent cell method. The transformant thus obtained was inoculated into L broth medium (pH 7.2) containing 20 μg/ml ampicillin and 10 μg/ml tetracycline followed by cultivation at 37° C. for 18 hours under shaking conditions. The transformants were collected from the culture by centrifugation and subjected to conventional alkali-SDS method to extract the recombinant DNA pCGPA/WT. The analysis of the pCGPA/WT by an automatic sequencer equipped with a fluorophotometer confirmed that it contained GPA/WT DNA, which termination codons were ligated to the 3'-terminus and was ligated to the downstream of a CMV promotor from the 5'- to 3'-termini.

The system using COS-1 (ATCC CRL-1650) as a host, which is a cell line derived from a monkey kidney, was used to express the DNA in the following Experiments 1 and 2. Since the system is for a transient expression, it has a disadvantage that DNAs introduced into transformants could not be stable over several days, and the transformants do not produce the desired polypeptides repeatedly. However, it is known that the number of copies of the desired DNA per cell temporally increases to 10⁵ when plasmid vectors having a replication origin derived from SV40 virus, such as the above mentioned pCDM8, are introduced into the COS-1 cells. With this point of view, the system has a merit that it quite easily analyzes the desired DNA-expression product.

EXPERIMENT 1-1(c)

Recombinant DNA Expression in COS-1 Cell

In accordance with the DEAE-dextran method reported by Frederick M. Ausubel et al. in "Current Protocols in Molecular Biology" (1987), chapters 9.2.1-9.2.3 and 9.2.5-9.2.6, published by John Wiley and Sons Inc., New York, U.S.A., the recombinant DNA pCGPA/WT in Experiment 1-1(b) was introduced into COS-1 cells for its expression. To each well of "3046", a plastic multiwell plate, with 6 wells of 3.5 cm diameter, commercialized by Becton Dickinson Labware, New Jersey, U.S.A., was added 2.5 ml of DME medium, containing 10 v/v % bovine fetal serum and 1.8×10⁵ COS-1 cells. The cells were cultured at 37° C. in a 5 v/v % CO₂ incubator overnight. After removing the culture supernatant by an aspirator and washing the remaining cells with DME medium containing 50 mM Tris-HCl buffer (pH 7.4), each well was charged with 2.5 ml of DME medium containing 2.8 μg/ml PCGPA/WT, 50 Mm Tris-HCl (pH 7.4), 0.4 mg/ml DEAE-dextran and 0.1 mM chloroquine, and incubated at 37° C. for 4 hours in a 5 v/v % CO₂ incubator. Thereafter, the culture supernatant was removed, and the remaining cells in each well were received with 2.5 ml of 10 mM phosphate buffered saline (hereinafter abbreviated as "PBS") containing 10 v/v % DMSO before incubating at ambient temperature for 2 minutes. After removing the supernatant and washing the remaining cells with DME medium containing 50 mM Tris-HCl (pH 7.4), each well was charged with 2.5 ml of "COS MEDIUM", commercialized by COSMO BIO CO. LTD., Tokyo, Japan, followed by cultivation at 37° C. for 3 days in a 5 v/v % CO₂ incubator to express the desired DNA. As a control, the same experiment was carried out using a plasmid vector, pCDM8.

After 3 days' cultivation, the multiwell plates with the cultures were subjected thrice to a treatment of freezing at -80° C. and thawing at ambient temperature to disrupt the cells. The whole cultures were transferred to centrifugal tubes and centrifuged to remove insoluble components after precipitated, followed by obtaining total soluble fractions, concentrating the fractions using membranes, and adjusting the volume of the total soluble fraction per well to give 0.5 ml for the following analyses.

EXPERIMENT 1-1(d)

Assay for L-asparaginase Activity

L-Asparaginase activity was expressed by the unit assayed as follows: Samples were placed in 1.5 ml-reaction tubes in 50 μl each and admixed with 200 μl of 50 mM phosphate buffer (pH 7.0) containing 1.4 mg/ml L-asparagine. After standing at 37° C. for 0, 1, 2, 4, 6 and 16 hours, L-aspartic acid in the reaction mixtures was quantified by an amino acid analyzer. In parallel, 1.0, 0.5 and 0.25 unit/ml dilutions of an L-asparaginase from Escherichia coli were provided and quantified for L-aspartic acid after incubating at 37° C. for 0 and one hour, and based on the increased amount of L-aspartic acid, a calibration curve was drawn. By plotting on the calibration curve the increased amounts of L-aspartic acid of the samples, the samples' L-asparaginase activities were estimated. The activity of samples with a lower activity was estimated based on that assayed after 2 hours or more incubation. One unit activity of L-asparaginase was defined as the amount that releases one umol of ammonia from L-asparagine per minute under the above conditions.

The total soluble fractions obtained in Experiment 1-1(c) were treated similarly as above, and expressed their activities as total L-asparaginase activities that were detected in the soluble fractions from 1.8×10⁵ COS-1 cells. As a result, the activity of the total soluble fraction in Experiment 1-1(c) was 0.083 unit, and the control gave no activity.

EXPERIMENT 1-1(e)

Western Blotting

An anti-L-asparaginase antibody was prepared as follows: An oligopeptide of a sequence Gly-Ser-Gly-Asn-Gly-Pro-Thr-Lys-Pro-Asp-Leu-Leu-Gln-Glu-Leu-Arg-Cys (SEQ ID NO:21) was synthesized chemically in a usual manner. Keyhole Limped Hemocyanin was linked to the C-terminus of the oligopeptide. The resultant was purified and used to immunize rabbits in a usual manner. The rabbits were immunized 6 times 2 weeks about, then the whole blood was collected and subjected to salting out with 50 w/v % ammonium sulfate to obtain an anti-L-asparaginase anti-serum.

In accordance with the method reported by U. K. Laemli et al. in "Nature", Vol.227, pp.680-685 (1970), 0.2 ml of the total soluble fraction in Experiment 1-1(c) was subjected to 12.5 w/v % SDS-polyacrylamide gel electrophoresis (hereinafter abbreviated as "SDS-PAGE"). The polypeptides migrated were transferred to a nitrocellulose membrane and subjected to Western blotting using the above anti-L-asparaginase anti-serum, in accordance with the method reported by H. Towbin in "Proceedings of the National Academy of Sciences of the U.S.A.", Vol.76, pp.4,350-4,354 (1979). For color development, alkaline phosphatase system was used. Comparing with the control and molecular weight markers, both the identification of bands specifically stained in the sample and the measurement of the molecular weight of each subunit of the L-asparaginase were carried out. The molecular weight markers used were bovine serum albumin (67 kDa), ovalbumin (45 Kda), soy bean trypsin inhibitor (20.1 kDa) and α-lactalbumin (14.4 kDa), and stained with amide black. The total soluble fraction in Experiment 1-1(c) gave no clear band.

EXPERIMENT 1-1(f)

Measurement of Molecular Weight on Gel Filtration

Two ml of the total soluble fraction in Experiment 1-1(c) was subjected to gel filtration column chromatography using "HILOAD SUPERDEX 200 COLUMN", with an inner diameter of 16 mm and a length of 60 cm, commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden, equilibrated with PBS. Based on the L-asparaginase activity of the eluted fractions, the molecular weight of the guinea pig wild-type L-asparaginase in a native form was examined. The molecular weight markers used were thyroglobulin (699 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (67 kDa) and ovalbumin (43 kDa). The peak of L-asparaginase activity in the eluted fractions was observed in a position corresponding to a molecular weight of about 300 kDa.

Since no clear band was detected by Western blotting, the molecular weight of the wild-type L-asparaginase in a dissociated form could not be detected, while the molecular weight in a native form was estimated to be about 300 kDa based on the result of gel filtration. The molecular weights of L-asparaginase in a native and dissociated form, purified from guinea pig L-asparaginase in serum, were respectively estimated to be about 190 kDa on gel filtration and about 43 kDa on DS-PAGE. As disclosed in Japanese Patent Kokai No.214,885/96 by the same applicant of the present invention, 3 partial amino acid sequences of a guinea pig L-asparaginase in serum were observed in a region of amino acids 10-236 in the sequence of guinea pig wild-type L-asparaginase. While, two consensus amino acid sequences essential for the expression of L-asparaginase activity, i.e., SEQ ID NOs:1 and 2, as proposed by E. Harms in "FEBS letters", Vol.285, pp.55-58 (1991) based on the results of experiments on L-asparaginase derived from Escherichia coli, correspond to the sequences of amino acids 16-19 and 114-118 in the amino acid sequence of the guinea pig wild-type L-asparaginase. In view of these and the results in Experiment 1-1, the present inventors estimated that the guinea pig wild-type L-asparaginase may require a region of amino acids about 1-400 in the amino acid sequence to express the activity. In Experiment 2-1, to examine the L-asparaginase activities of C-terminal defective mutants as homologues of the guinea pig wild-type L-asparaginase, the expression products of DNA homologues from a guinea pig were tested for properties and features.

EXPERIMENT 1-2

Expression of Human Wild-type DNA

A human wild-type DNA encoding L-asparaginase was prepared according to the method in Japanese Patent Kokai No.214,855/96 by the same applicant of the present invention. The DNA had the nucleotide sequence of SEQ ID NO:16. Hereinafter, a DNA having a polypeptide-encoding region in SEQ ID NO:16, i.e., a sequence of nucleotides 93-1,811 in SEQ ID NO:16, was named "HA/WT DNA", and a polypeptide, as the expression product of HA/WT DNA, having the amino acid sequence of SEQ ID NO:50, may be called "human wild-type L-asparaginase". SEQ ID NO:18 shows the nucleotide sequence of GPA/WT DNA and the amino acid sequence (SEQ ID NO:50) encoded thereby.

Except for the template and the sense- and anti-sense-primers, PCR was performed under the same conditions as used in Experiment 1-1(b). As a template, the human wild-type DNA in Experiment 1-2 was used. As a sense- and anti-sense-primers, oligonucleotides with sequences of 5'-AATCTCGAGCCACCATGGCGCGCGCG GTG-3' (SEQ ID NO:22) and 5'-CTGCGGCCGCTTATCAGACACCAGGCAGCAC-3' (SEQ ID NO:23) were respectively used. The DNA thus amplified was continuously treated with the same method as used in Experiment 1-1(b) to prepare a recombinant DNA, "pCHA/WT". After sequencing, the pCHA/WT was introduced into COS-1 cells and expressed followed by analyzing the expression product similarly as in Experiment 1-1.

In contrast to the guinea pig wild-type L-asparaginase, the experiment system could not detect the human wild-type L-asparaginase activity. It was presumably due to that the human wild-type L-asparaginase had a lower specific activity than that of the guinea pig wild-type one, and this forced to examine the properties of expression products by DNA homologues from human in Experiment 2-2.

EXPERIMENT 2

Expression of DNA Homologue

EXPERIMENT 2-1

Expression of DNA Homoloque Originating From Guinea Pig

A termination codon was replaced for the nucleotide sequence in a specific position of the guinea pig wild-type DNA to obtain a DNA homologue: A DNA was obtained by PCR method by replacing a termination codon for a codon of the nucleotides 1,090-1,092 or 1,012-1,014 in SEQ ID NO:17. Except for the nucleotide sequence of anti-sense primer, PCR was performed under the same conditions as used in Experiment 1-1(b). As an anti-sense primer, an oligonucleotide with a sequence of 5'-CTGCGGCCGCTTATCATGCCGTGGGCAGTGT-3' (SEQ ID NO:24) or 5'-CTGCGGCCGCTTATCAGCCCAACACGTAGGA-3' (SEQ ID NO:25) was used to prepare the two-types of DNAs. The amplified DNAs were treated similarly as in Experiment 1-1(b) to obtain recombinant DNAs, "pCGPA/D364stp" and "pCGPA/L338stp". By sequencing similarly, it was confirmed that pCGPA/D364stp and pCGPA/L338stp had DNAs, encoding the sequences of amino acids 1-363 and 1-337 in the guinea pig wild-type L-asparaginase, respectively, and had a termination codon at their 3'-termini free of intervening sequences. Hereinafter, the polypeptide-encoding regions of the DNAs are respectively named "GPA/D364stp DNA" and "GPA/L338stp DNA". GPA/D364stp DNA and GPA/L338stp DNA were ligated in the downstream of a CMV promoter in the direction from the 5'- to 3'-termini. The DNAs expression products may be named "guinea pig L-asparaginase homologues".

The above recombinant DNAs were introduced into COS-1 cells and examined similarly as in Experiment 1-1. As controls, pCGPA/WT and pCDM8 in Experiment 1-1(b) were similarly treated and examined. Table 1 shows the results.

                  TABLE 1                                                          ______________________________________                                                    L-asparaginase                                                                             Molecular                                                  activity weight Molecular weight                                              Recombinant DNA (unit) (kDa) *1 (kDa) *2                                     ______________________________________                                         pCGPA/WT   0.083       --       about 300                                        pCGPA/D364stp 0.228 about 40 about 140                                         pCGPA/L338stp N.D. *3 about 40 --                                              pCDM8 N.D. *3 -- --                                                          ______________________________________                                          Note: The symbols "*1", "*2" and "*3" mean that the value was determined       by Western blotting, the value was determined by gel filtration, and the       activity was not detected, respectively.                                 

As shown in Table 1, the activities of the expression products of GPA/WT DNA and GPA/D364stp DNA were detected, but not for GPA/L338stp DNA. These results suggest that a region of amino acids 1-363 in the guinea pig wild-type L-asparaginase may be enough for sufficiently expressing the L-asparaginase activity. This amino acid sequence, amino acids 1-363 in the guinia pig wild-type, is SEQ ID NO:4, and a nucleotide sequence which encodes the amino acid sequence is SEQ ID NO:10. The amino acid sequence of the guinea pig wild-type L-asparaginase is SEQ ID NO:5.

EXPERIMENT 2-2

Expression of DNA Homoloque Originating from Human

DNA homologues were prepared by replacing specific codons in the human wild-type DNA with termination codons or codons for different amino acids: The DNA homologues were prepared by replacing termination codons for the nucleotides 1096-1098 in SEQ ID NO:18 by applying PCR method. Except for the template and the sense- and anti-sense-primers, PCR was performed under the same conditions as used in Experiment 1-1(b). As a template, the human wild-type DNA in Experiment 1-2 was used. As a sense- and anti-sense-primers, the oligonucleotides with sequences of 5'-AATCTCGAGCCACCATGGCGCGCGCGGTG-3' (SEQ ID NO:25) and 5'-CTGCGGCCGCTCATTACACCGAGGGTGGCGT-3' (SEQ ID NO:26) were respectively used. The amplified DNA was treated similarly as in Experiment 1-1 to obtain a recombinant DNA, "pCHA/E366stp", and sequenced. It was confirmed that pCHA/E366stp contained a DNA encoding amino acids 1-365 in SEQ ID NO:16 and a termination codon at the 3'-terminus free of intervening sequences. The polypeptide-encoding region was named "HA/E366stp DNA", hereinafter. HA/E366stp DNA was ligated to the downstream of a CMV promotor in the direction from the 5'- to 3'-termini.

To change specific codons in DNAs into ones for different amino acids, the over lap extension method reported by Robert M. Horton et al. in "Methods in Enzymology", Vol.217, pp.270-279 (1993), published by Academic Press, Inc., San Diego, U.S.A., was used. The method is summarized in FIG. 1 and explained as follows: First, mutagenic primers A and B, where the nucleotides to be mutagenized were substituted by desired different ones complementary to one another, were prepared. The mutagenic primer A was a sense strand, and the mutagenic primer B was an anti-sense strand. A set of 5'- and 3'-terminal primers, which amplify the whole region of the desired DNA, were prepared, and they were respectively a sense- and anti-sense-strands. Second, conventional PCR was performed using the 5'-terminal primer, the mutagenic primer A, and as a template, a DNA with the original nucleotide sequence. In parallel, another PCR as was performed using the same DNA as a template, the 3'-terminal primer, and the mutagenic primer B. These two PCRs were named "first step PCRs". Third, two DNAs amplified in the first step PCRs were mixed with the 5'- and 3'-terminal primers as used in the first step PCRs followed by performing PCR as a second step PCR. The two DNA fragments amplified in the first step PCRs were used as primers and templates to generate mutagenized DNAs, while the 5'- and 3'-terminal primers were used as primers to amplify the mutagenized DNAs. By this method, DNAs into which were introduced 7 types nucleotide substituents, i.e., 7 DNA homologues were prepared. The 7 types nucleotide substituents and consequent changes of the encoded amino acid sequences are summarized in Table 2. The template DNA and mutagenic primers A and B used to prepare the 7 DNA homologues were summarized in Table 3. The 5'- and 3'-terminal primers were respectively equal to the sense- and anti-sense-primers as used to prepare pCHA/E366stp in Experiment 2-2.

                                      TABLE 2                                      __________________________________________________________________________     DNA homologue                                                                          Recombinant DNA                                                                          Nucleotide substitution (upper line) and                                                   consequential change of amino acid (lower                          line) *                                                      __________________________________________________________________________     HA/MUT1 DNA                                                                            pCHA/MUT1 C894G, A902G, G952A, G953A and G1096T                                                      H298Q, Q301R, G318N and E366stp                    HA/MUT2 DNA  pCHA/MUT2      C894G, A902G and G1096T                                                        H298Q, Q301R and E366stp                           HA/MUT3 DNA  pCHA/MUT3      C894G, G952A, G953A and G1096T                                                 H298Q, G318N and E366stp                           HA/MUT4 DNA  pCHA/MUT4      A902G, G952A, G953A and G1096T                                                 Q301R, G318N and E366stp                           HA/MUT5 DNA  pCHA/MUT5      C894G and G1096T                                                               H298Q and E366stp                                  HA/MUT6 DNA  pCHA/MUT6      A902G and G1096T                                                               Q301R and E366stp                                  HA/MUT7 DNA  pCHA/MUT7      G952A, G953A and G1096T                                                        G318N and E366stp                                __________________________________________________________________________      *) Numbers in the upper lines in each column mean a nucleotide number in       SEQ ID NO: 18. Numbers in the lower lines in each column means an amino        acid residue number in SEQ ID NO: 18. Alphabets on the left and right of       the numbers in the upper lines show nucleotides before and after the           nucleotide substitution, respectively. Alphabets on the left and right of      the numbers in the lower lines show amino acids before and after the           nucleotide  substitution, respectively. The symbol "stp" means that a          termination condon was substituted for a codon in the wildtype DNA. Names      for the 7 DNA homologues and the recombinant DNAs containing the DNA           homologues are shown in parallel.                                        

                                      TABLE 3                                      __________________________________________________________________________     DNA homologue                                                                          Template DNA                                                                           Nucleotide sequences of mutagenic primers A (upper                                                            line) and B (lower line)        __________________________________________________________________________                     *                                                              HA/MUT1 DNA                                                                            pCHA/MUT7                                                                              the same as used for HA/MUT2 DNA preparation                                                   the same as used for HA/MUT2 DNA preparati                                                on                                    HA/MUT2 DNA    pCHA/E366stp   5'-CCCCcGGAGGCAcTGGGT-3' (SEQ ID NO: 27)                                                  5'-ACCCAgTGCCTCCgGGGG-3' (SEQ                                                  ID NO: 28)                            HA/MUT3 DNA    pCHA/MUT7      the same as used for HA/MUT5 DNA preparati                                                on                                                                  the same as used for HA/MUT5 DNA preparati                                                on                                    HA/MUT4 DNA    pCHA/MUT7      the same as used for HA/MUT6 DNA preparati                                                on                                                                  the same as used for HA/MUT6 DNA preparati                                                on                                    HA/MUT5 DNA    pCHA/E366stp   5'-CCCCTGGAGGCAcTGGGT-3' (SEQ ID NO: 29)                                                  5'-ACCCAgTGCCTCCAGGGG-3' (SEQ                                                  ID NO: 30)                            HA/MUT6 DNA    pCHA/E366stp   5'-CCCCcGGAGGCAGTGGGT-3' (SEQ ID NO: 31)                                                  5'-ACCCACTGCCTCCgGGGG-3' (SEQ                                                  ID NO: 32)                            HA/MUT7 DNA    pCHA/E366stp   5'-GACGttGGCTCCCGCCAT-3' (SEQ ID NO: 33)                                                  5'-ATGGCGGGAGCCaaCGTC-3' (SEQ                                                  ID NO: 34)                          __________________________________________________________________________      Note: Small letters mean nucleotides which were substituted for those in       human wildtype DNA.                                                      

The obtained DNA homologues from human were treated similarly as in Experiment 1-1 to obtain recombinant DNAs "pCHA/MUT1", "pCHA/MUT2", "pCHA/MUT3", "pCHA/MUT4", "pCHA/MUT5", "pCHA/MUT6", "pCHA/MUT7". The expression products of the DNA homologues, obtained in Experiment 2-2, may be named "human L-aspraginase homologues", hereinafter. After sequencing, these DNA homologues were introduced into COS1 cells, followed by expression and assay. As controls, pCHA/WT obtained in Experiment 1-2 and pCDM8 were treated and examined. Signal intensities of bands, detected by Western blotting, were evaluated by densitometry to compare quantitatively the expressed product. The results were in Table 4.

                  TABLE 4                                                          ______________________________________                                                   L-asparaginase                                                                            Molecular        Molecular                                  Recombinant activity weight Quantity weight                                    DNA (unit) *1 (kDa) *2 *3 (kDa) *4                                           ______________________________________                                         pCHA/WT   N.D.       --        --     --                                         pCHA/E366stp N.D. about 40 2.3 --                                              pCHA/MUT1 0.021 about 40 0.4 about 140                                         pCHA/MUT2 0.031 about 40 0.9 about 140                                         pCHA/MUT3 0.009 about 40 0.1 about 140                                         pCHA/MUT4 N.D. about 40 0.2 --                                                 pCHA/MUT5 0.006 about 40 1.2 about 140                                         pCHA/MUT6 N.D. about 40 1.9 --                                                 pCHA/MUT7 N.D. about 40 0.2 --                                                 pCDM8 N.D. -- -- --                                                          ______________________________________                                          Note: The symbols "*1", "*2", "*3" and "*4" mean the activity was not          detected, the value was determined by Western blotting, the value              indicates the signal intensity of the band detected on Western blotting        and quantified by densitometry, and the value was determined by gel            filtration, respectively.                                                

The results in Table 4 indicate that human L-asparaginases both in the wild-type and in the C-terminal defected mutant, i.e., the expression product of HA/E366stp DNA, as the one of the homologues, had a lower specific activity than that from guinea pigs. In addition, these results indicate that the specific activity of L-asparaginases among those of point mutants, which some of the amino acids inherent to the human wild-type L-asparaginase were substituted by different ones, increased to a detectable level. The human DNA homologues such as HA/MUT1, HA/MUT2, HA/MUT3 and HA/MUT5, which the expression products gave a detectable level of activity, have SEQ ID NOs:11 to 14, respectively, and encoding SEQ ID NOs:6 to 9, respectively.

Based on the results in the above experiments, the present inventors found that polypeptides from mammal may require the amino acid sequence of SEQ ID NO:3 (where the symbol "Xaa" meant "glutamine" or "arginine") to express a detectable level of L-asparaginase activity in the expression and assay systems in Experiments 1 and 2, in addition to conventionally known as such amino acid sequences of SEQ ID NOs:1 and 2. The animo acid sequence of the guinea pig wild-type L-asparaginase contains the SEQ ID NO:3 in the region the amino acids 298-302. Examples of such polypeptides, having all the amino acid sequences of SEQ ID NOs: 1 to 3, include those having SEQ ID NOs:4 and 5 from guinea pigs and those having SEQ ID NOs:6 to 9 from human.

Based on the above findings, the present inventors invented the polypeptides having L-asparaginase activity. The following examples explain the present invention, and the techniques used therein are conventional ones used in the art, and of course, they are not restrictive to the present invention:

EXAMPLE A-1

Polypeptides Having L-asparaginase Activity

EXAMPLE A-1(a)

Preparation of Transformant

Ten μl of 10×PCR buffer, one μl of 25 mM dNTP mix, one ng of the recombinant DNA pCGPA/WT DNA obtained in Experiment 1-1 as a template, and an adequate amount of oligonucleotides as a sense- and anti-sense-primers synthesized chemically based on the 5'- and 3'-terminal sequences of GPA/WT DNA were placed in 0.5 ml reaction tube. The mixture was mixed with sterilized distilled water to give a total volume of 99.5 μl, and 0.5 μl of 2.5 units/μl AmpliTaq DNA polymerase were further added. The sequence of the sense primer was 5'-GCGAATTCATGGCGCGCGCATCA-3' (SEQ ID NO:35) which was a nucleotide sequence obtained by adding a cleavage site by a restriction enzyme, Eco RI, to the upstream of the 5'-terminus of GPA/WT DNA. The sequence of the anti-sense primer was 5'-GCAAGCTTTCAGATGGCAGGCGGCAC-3'(SEQ ID NO:36), which was complementary to a nucleotide sequence prepared by adding a termination codon to the 3'-terminus of GPA/WT DNA and then adding a cleavage site by a restriction enzyme, Hin dIII, to the downstream. The above mixture was subjected to 40 cycles of successive incubations at 94° C. for one min, at 55° C. for one min, and 72° C. for 3 min to perform PCR. By cleaving the amplified DNA by restriction enzymes Eco RI and Hin dIII, a Eco RI-Hin dIII fragment with a length of about 1.7 kbp was obtained. Twenty-five ng of the DNA was mixed with 10 ng of plasmid vector "pKK223-3", commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden, which had been cleaved by restriction enzymes Eco RI and Hin dIII, and then mixed with the solution I in "LIGATION KIT VERSION 2" commercialized by Takara Shuzo Inc., Tokyo, Japan, in an equal volume of the DNA mixture, followed by incubation at 16° C. for 2 hours to obtain a replicable recombinant DNA, "pKGPA/WT".

The recombinant DNA pKGPA/WT was introduced into an Escherichia coli strain "JM105" by the competent cell method. The resulting transformant "J-GPA/WT" was inoculated to L broth medium (pH 7.2) containing 50 μg/ml ampicillin and cultured at 37° C. for 18 hours under shaking conditions. The transformants collected by centrifugation from the culture were subjected to a conventional alkali-SDS method to extract the recombinant DNA pKGPA/WT. As shown in FIG. 2, analysis using an automatic sequencer equipped with a fluorophotometer revealed that GPA/WT DNA of SEQ ID NO:17 ligated to the downstream of a Tac promotor in the direction from the 5'- to 3'-termini. In addition, it was confirmed that a termination codon was ligated to the downstream of GPA/WT DNA without intervening sequences.

EXAMPLE A-1(b)

Production of Polypeptide

The transformant J-GPA/WT was inoculated into L broth medium (pH 7.2), containing 50 μg/ml ampicillin, and cultured at 37° C. for 18 hours under shaking conditions to obtain a seed culture. Eighteen L of a fresh preparation of the same medium was placed in a 30-L jar fermenter, inoculated with one v/v % of the seed culture, and cultured at 37° C. under aeration-agitation conditions. A portion of the culture was placed in a cuvette with 1-cm in thickness, incubated until the absorbance at a wavelength of 650 nm reached to about 1.5, admixed with IPTG to give a final concentration of 0.1 mM, and incubated for 5 hours. The cells centrifugally collected from the culture were suspended in a mixture solution (pH 7.2) containing 139 mM NaCl, 7 mM Na₂ HPO₄ and 3 mM NaH₂ PO₄, and supersonicated to disrupt the cells, followed by centrifuging the resultant to obtain a supernatant.

Ammonium sulfate was added to the supernatant under ice-chilling conditions to give a concentration of 50 w/v % and then dissolved to homogeneity. After standing for several minutes, the precipitates were collected by centrifugation, dissolved in 20 mM Tris-HCl buffer (pH 8.0), and dialyzed against a fresh preparation of the same buffer followed by applying the dialyzed solution to "Q SEPHAROSE FF COLUMN", commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden, equilibrated with the same buffer. After washing sufficiently with the same buffer, the column was fed with a linear gradient buffer of NaCl increasing from 0 M to 0.5 M in 20 mM Tris-HCl buffer (pH 8.0). The fractions eluted at about 0.1-0.3 M NaCl were collected, and the solvent was replaced with 10 mM sodium-phosphate buffer (pH 7.5) while concentrating with membranes. The concentrated solution was then applied to "L-ASPARAGINE AGAROSE", commercialized by Sigma Chemical Co., St. Louis, U.S.A., equilibrated with the same buffer. After washing with the same buffer, 10 mM sodium phosphate buffer (pH 7.5) containing 0.5 M NaCl was fed to the column for elution. The eluted fractions were pooled and concentrated by using a membrane. The concentrate was applied to "HILOAD SUPERDEX 200 COLUMN", commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden, equilibrated with Tris-HCl buffer (pH 8.0) containing 10 v/v % glycerol, and eluted from the column. The eluted fractions, containing substances with a molecular weight of about 300 kDa, were collected to obtain a purified polypeptide with a purity of 90% or more in a yield of about 0.1 mg/ml culture.

EXAMPLE A-1(c)

Physicochemical Property of Polypeptide

The purified polypeptide in the above was analyzed to determine the physicochemical properties: The molecular weight of the purified polypeptide in a native form was determined by gel filtration similarly as in Experiment 1-1(e). The peak for L-asparaginase activity of the eluted fractions was found at a position corresponding to a molecular weight of about 300 kDa. The molecular weight of the purified polypeptide in a dissociated form was determined by SDS-PAGE as used in Experiment 1-1(e). The main band was observed at a position corresponding to a molecular weight of about 50±10 kDa. The results indicate that the purified polypeptide exists in a multimer as a native form. Considering errors in measurement by the above methods and the fact that all the known L-asparaginases from Escherichia Coli etc., other than mammal, exist in a tetrameric form, it can be estimated that the purified polypeptide exists in a tetrameric form. The method as used in Experiment 1-1(d) confirmed that the purified polypeptide has an L-asparaginase activity.

EXAMPLE A-2(a)

Preparation of Transformant

FIG. 3 summarizes the procedures to prepare transformants. PCR was performed under the same conditions as used in Example A-1(a) except for the nucleotide sequences of a sense- and anti-sense-primers. As the sense- and anti-sense-primers, oligonucleotides with the nucleotide sequences of 5'-GTGAATTCGGAGGTTCAGATGGCGCGCGCATCA-3' (SEQ ID NO:37) and 5'-CTGCGGCCGCTCAGATGGCAGGCGGCAC-3' (SEQ ID NO:38) were respectively used. The DNA thus amplified was cleaved by restriction enzymes Eco RI and Not I to obtain an about 1.7 kbp Eco RI-Not I fragment. Seventy ng of the DNA fragment was mixed with 50 ng of a plasmid vector, "pBPV", commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden, cleaved in advance by restriction enzymes Xho I and Not I, and 25 ng of each of 4 oligonucleotides as linkers with nucleotide sequences of 5 '-TCGAGCCACCATGAAGTGTTCGTGGGTTATT-3'(SEQ ID NO:39), 5'-TTCTTCCTGATGGCCGTAGTGACAGGAGTG-3'(SEQ ID NO:40), 5'-AATTCACTCCTGTCACTACGGCCATCAGGA-3'(SEQ ID NO:41), and 5'-AGAAAATAACCCACGAACACTTCATGGTGGC-3'(SEQ ID NO:42). The oligonucleotides for linkers were synthesized in a usual manner and used after reacted with T4 polynucleotide kinase, commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden, and purified by ethanol-precipitation. To the DNA mixture was added the solution I in "LIGATION KIT VERSION 2", commercialized by Takara Shuzo, Tokyo, Japan. The mixture was incubated at 16° C. for 2 hours to obtain a replicable recombinant DNA "pBIgGPA/WT".

The recombinant DNA pBIgGPA/WT was introduced into an Escherichia coli HB101 strain by the competent cell method. The transformant thus obtained was inoculated into L broth medium (pH 7.2) containing 50 μg/ml ampicillin followed by cultivation at 37° C. for 18 hours under shaking conditions. The transformants, collected by centrifuging the resulting culture, were subjected to a conventional alkali-SDS method to extract the recombinant DNA pBIgGPA/WT. The nucleotide sequence analysis using an automatic sequencer confirmed that the recombinant DNA pBIgGPA/WT had the structure in FIG. 4: A DNA encoding a polypeptide containing a signal sequence for immunoglobulin secretion, as shown by D. F. Stern et al. in "Science", Vol.235, pp.321-324 (1984), i.e., "Ig sec DNA" was ligated to the downstream of a region for transcriptional regulation, comprising an enhancer derived from long terminal repeats of Moloney Mouse Sarcoma Virus (Emsv), and a promotor derived from Mouse metallothionein I gene (Pmti). Furthermore, GPA/WT DNA was ligated in the same frame to the downstream of the Ig sec DNA in the direction from the 5'- to 3'-termini of GPA/WT DNA. It was also confirmed that a termination codon exists in the 3'-terminus of GPA/WT DNA without intervening sequences.

The recombinant DNA pBIgGPA/WT was introduced into a cell line C127 (ATCC CRL-1616), derived from a mouse, by using a lipofectin® reagent commercialized by Life Technologies, Inc., Gaitherburg, U.S.A., according to the attached protocol. The transformants with the recombinant DNA were selected based on the lack of proliferation-regulatory ability, i.e., focus-forming ability, as a first selection. The cells around those containing foci were collected using sterilized filter papers and subjected to a conventional limiting dilution method to form single cells which were then selected depending on the productivity of L-asparaginase, as final selection. Thus, a transformant, "C-GPA/WT", was obtained.

EXAMPLE A-2(b)

Production of Polypeptide

The transformant C-GPA/WT was inoculated into a well of "3046", a plastic multiwell plate with 6 wells, 3.5 cm in diameter, commercialized by Becton Dickinson Labware, New Jersey, U.S.A., with DME medium containing 10 v/v % bovine fetal serum, and cultured to be confluent as a seed culture. Some of the cells, scraped by treatment with trypsin, were inoculated as seed cells into each of the multiwell plates which were charged with the fresh preparation of the same medium and cultured. After repeating manipulations similarly as in the above and with scale up to increase the cell number, the cells were subjected to a conventional continuous culture using 50 of 150 cm² culture flasks. The resulting culture supernatants of a volume of 100 l was collected and treated with similar methods for treating the supernatant from the cell-disruptants in Example A-1(b): salting out with ammonium sulfate, the chromatography of the solution of the precipitates using Q SEPHAROSE FF COLUMN, the chromatography of the eluted fractions using L-ASPARAGINE AGAROSE, and the chromatography of the eluted fractions using HILOAD SUPERDEX 200 COLUMN. Consequently, a purified polypeptide with a purity of 90% or more was obtained in a yield of about one pg/ml-culture.

EXAMPLE A-2(c)

Physicochemical Property of Polypeptide

By testing similarly as in Example A-1(c), it was confirmed that the purified polypeptide thus obtained had equivalent physicochemical properties with the that obtained in

EXAMPLE A-1(b). EXAMPLE A-3(a)

Preparation of Transformant

PCRs were performed under the same conditions in Example A-1(a) except for the template and the sense- and anti-sense-primers. The DNA thus obtained were treated similarly as in Example A-1(a) to prepare recombinant DNAs, "pKGPA/D364stp", "pKHA/MUT1", "pKHA/MUT2", "pKHA/MUT3" and "pKHA/MUT5". Table 5 summarizes template DNAs and nucleotide sequences of a sense-and anti-sense-primers which were used to prepare the each recombinant DNAs. By sequencing similarly as in Example A-1(a), the structures of these recombinant DNAS were confirmed as shown in FIGS. 5 to 9.

                                      TABLE 5                                      __________________________________________________________________________     Recombinant DNA                                                                          Template DNA                                                                           Nucleotide sequences of sense (upper line)                                                        and anti-sense (lower 1ine) primers       __________________________________________________________________________                       *                                                            pKGPA/D364stp                                                                            pCGPA/D364stp                                                                          5'-GCGAATTCATGGCGCGCGCATCA-3'                                                                            (SEQ ID NO: 35)                                                         5'-GCAAGCTTTCATGCCGTGGGCAGTGT-3'(SEQ                                                  ID NO: 43)                           pKHA/MUT1          pCHA/MUT1       5'-GCGAATTCATGGCGCGCGCGGTG-3'(SEQ ID                                                  NO: 44)                                                                 5'-GCAAGCTTTCACACGAGGGTGGCGT-3'(SEQ                                                   ID NO: 45)                           PKHA/MUT2          pCHA/MUT2       the same as used for pKHA/MUT1                                                        preparation                                                             the same as used for pKHA/MUT1                                                        preparation                          pKHA/MUT3          pCHA/MUT3       the same as used for pKHA/MUT1                                                        preparation                                                             the same as used for pKHA/MUT1                                                        preparation                          pKHA/MUT5          pCHA/MUT5       the same as used for pKHA/MUT1                                                        preparation                                                             the same as used for PKHA/MUT1                                                        preparation                        __________________________________________________________________________      *) Italics in the upper line in each column mean the 5terminal nucleotide      sequence of a DNA encoding Lasparaginase, and those in the lower line mea      the complementary sequence to the 3terminus of the DNA, wherein the            Lasparaginese originates from a guinea  pig or human.                    

The recombinant DNAs were treated according to the methods as in Example A-1(a) to obtain transformants, "J-GPA/D364stp", "J-HA/MUT1", "J-HA/MUT2", "J-HA/MUT3" and "J-HA/MUT5".

EXAMPLE A-3(b)

Production of Polypeptide

The transformants obtained in Example A-3(a) were treated according to the methods similarly as in Example A-1(b): cultivation, disrupting the resulting cells, the precipitations of the cell-disruptants with ammonium sulfate, the chromatography of the precipitate solutions using Q SEPHAROSE FF COLUMN, and the chromatography of the eluted fractions using L-ASPARAGINE AGAROSE in that order. The eluted fractions thus obtained were concentrated using membranes similarly as in Example A-1(b) followed by subjecting the chromatography using HILOAD SUPERDEX 200 COLUMN to collect the eluted fractions with a molecular weight of about 140 kDa. Each system yielded the purified polypeptide with a purity of 90% or more in a yield of about 0.1 mg/ml-culture. These purified polypeptides were analyzed by the methods as in Example A-1(c) to examine their physicochemical properties. Table 6 shows the results combined with those in Example A-1(c).

                  TABLE 6                                                          ______________________________________                                         Transformant,                                                                             Molecular  Molecular                                                  producing the weight weight L-asparaginase                                     polypeptide (kDa) *1 (kDa) *2 activity                                       ______________________________________                                         J-GPA/WT   about 300  about 50 ± 10                                                                          +                                               J-GPA/D364stp about 140 about 40 +                                             J-HA/MUT1 about 140 about 40 +                                                 J-HA/MUT2 about 140 about 40 +                                                 J-HA/MUT3 about 140 about 40 +                                                 J-HA/MUT5 about 140 about 40 +                                               ______________________________________                                          Note) The symbols "*1" and "*2" mean that the value was determined by gel      filtration, and the value was determined by SDSPAGE, respectively.       

Table 6 indicates that all of the present polypeptides, expressed in Escherichia Coli and purified, expressed an L-asparaginase activity. Furthermore, table 6 indicates the that the polypeptides formed tetramers.

Example A-4(a)

Preparation of Transformants

PCRs were performed under the same conditions in Example A-1(a) except for the template and the sense- and anti-sense-primers. DNAs thus obtained were ligated with the same linkers as used in Example A-2(a) under the same conditions as in Example A-2(a) to obtain recombinant DNAs, "pBIgGPA/D364stp", "pBIgHA/MUT1", "pBIgHA/MUT2", "pBIgHA/MUT3" and "pBIgHA/MUT5". Table 7 summarizes template DNAs and nucleotide sequences of sense- and anti-sense-primers which were used to prepare the each recombinant DNAs. By sequencing similarly as in Example A-1(a), the structures of these recombinant DNAs were confirmed as shown in FIGS. 10 to 14.

                                      TABLE 7                                      __________________________________________________________________________     Recombinant DNA                                                                          Template DNA                                                                           Nucleotide seguences of sense (upper line)                                                        and anti-sense (lower line) primers       __________________________________________________________________________                       *                                                            pBIgGPA/D364stp                                                                          pCGPA/D364stp                                                                          5'-GTGAATTCGGAGGTTCAGATGGCGCGCGCATCA-3'                                                                   (SEQ ID NO: 37)                                                        5'-CTGCGGCCGCTCATGCCGTGGGCAGTG-3'(SEQ                                                   ID NO: 46)                         pBIgHA/MUT1        pCHA/MUT1       5'-CTGAATTCGGAGGTTCAGATGGCGCGCGCGGTG-                                                  3'(SEQ ID NO: 47)                                                      5'-CTGCGGCCGCTCACACCGAGGGTGGCG-3'(SEQ                                                   ID NO: 48)                         pBIgHA/MUT2        pCHA/MUT2       the same as used for pBIgHA/MUT1                                                       preparation                                                            the same as used for pBIgHA/MUT1                                                       preparation                         pBIgHA/MUT3        pCHA/MUT3       the same as used for pBIgHA/MUT1                                                       preparation                                                            the same as used for pBIgHA/MUT1                                                       preparation                         pBIgHA/MUT5        pCHA/MUT5       the same as used for pBIgHA/MUT1                                                       preparation                                                            the same as used for pBIgHA/MUT1                                                       preparation                       __________________________________________________________________________      Note)                                                                          *: Italics in the upper line in each column mean the 5terminal nucleotide      sequence of a DNA encoding Lasparaginase, and those in the lower line mea      the complementary seguence to the 3terminus of the DNA, wherein the            Lasparaginese originates from a guinea pig or human.                     

The recombinant DNAs thus obtained were treated similarly as in Example A-2(a) to obtain transformants, "C-GPA/D364stp", "C-HA/MUT1", "C-HA/MUT2", "C-HA/MUT3" and "C-HA/MUT5".

Example A-4(b)

Production of Polypeptide

The transformants obtained in Example A-4(a) were cultured according to the methods as in Example A-2(b), and the resulting culture supernatants were treated with similar methods for treating the supernatants from the cell-disruptants in Example A-1(b): the precipitations of culture supernatants with ammonium sulfate, the chromatography of the precipitate solutions using Q SEPHAROSE FF COLUMN, and the chromatography of the eluted fractions using L-ASPARAGINE AGAROSE in that order. The eluted fractions thus obtained were concentrated using membranes similarly as in Example A-1(b) followed by subjecting the chromatography using HILOAD SUPERDEX 200 COLUMN to collect the eluted fractions with a molecular weights of about 140 kDa. Each of these systems yielded the purified polypeptide with a purity of 90% or more in a yield of about one μg/ml-culture. These purified polypeptides were analyzed by the methods as in Example A-1(c) to examine their physicochemical properties. Table 8 shows the results combined with those in Example A-3.

                  TABLE 8                                                          ______________________________________                                         The polypeptide-                                                                 producing Molecular weight Molecular weight L-asparaginase                     transformant (kDa) *1 (kDa) *2 activity                                      ______________________________________                                         J-GPA/WT  about 300   about 50 ± 10                                                                           +                                              J-GPA/D364stp about 140 about 40 +                                             J-HA/MUT1 about 140 about 40 +                                                 J-HA/MUT2 about 140 about 40 +                                                 J-HA/MUT3 about 140 about 40 +                                                 J-HA/MUT5 about 140 about 40 +                                               ______________________________________                                          Note) The symbols "*1" and "*2" mean that the value was determined by gel      filtration, and the value was determined by SDSPAGE, respectively.       

Table 8 indicates that all of the present polypeptides, expressed in mammalian cells and purified, expressed an L-asparaginase activity. Furthermore, table 8 indicates the polypeptides formed tetramers.

As shown in above Example A, each of the polypeptides according to the present invention expresses an L-asparaginase activity. Therefore, the present agent for susceptive diseases hydrolyze L-asparagine in patients to exert therapeutic and preventive effects on L-asparaginase-susceptive diseases when administered to human. The wording "susceptive diseases" as referred in the present specification means diseases in general which are caused by the existence of tumor cells dependent on L-asparagine: For example, leukemias such as acute leukemia, an acute transformation of chronic leukemia and T-lymphocytic leukemia, and malignant tumors such as Hodgkin's diseases and non-Hodgkin's diseases. The present agent for susceptive diseases possesses thus the uses as anti-tumor agents for treating and/or preventing such susceptive diseases as above. Although it varies dependently on the types of agents used for such purposes and susceptive diseases to be treated, the present agent is generally processed into an agent in the form of a liquid, a paste or a solid which contains the polypeptides in an amount of 0.000001-100 w/w %, preferably, 0.0001-100 w/w %, on a dry solid basis.

The present agent can be used intact or processed into compositions by mixing with one or more selected from the group consisting of physiologically-acceptable carriers, excipients, solvents, buffers and stabilizers, and further, if necessary, other biologically-active substances and other agents. For example, "Iyakuhin-Tenkabutsu-Jiten (The Dictionary of Pharmaceutical Excipients)" (1994), edited by Japan Pharmaceutical Excipients Council, Tokyo, Japan, published by Yakujinippo LTD., Tokyo, Japan and "Iyakuhin-Tenkabutsu-Jiten-Tsuiho 1995 (Suppliment for The Dictionary of Pharmaceutical Excipients)" (1995), edited by Japan Pharmaceutical Excipients Council, Tokyo, Japan, published by Yakujinippo LTD., Tokyo, Japan, mention the embodiments of such carriers, excipients, solvents, buffers and stabilizers. Examples of such other biologically-active substances and other agents include interferon-α, interferon-β, interferon-γ, interleukin 1, interleukin 2, interleukin 3, TNF-α, TNF-β, GM-CSF, carboquone, cyclophosphamide, aclarbicin, thiotepa, busulfan, ancitabine, cytarabine, fluorouracil, 5-fluoro-1-(tetrahydro-2-furyl)uracil, methotrexate, actinomycin D, chromomycin A3, daunorubicin, doxorubicin, bleomycin, mercaptopurine, prednisolone, mitomycin C, vincristine, vinblastine, radio gold colloidal, Krestin®, picibanil, lentinan and Maruyama vaccine.

The present agent for susceptive diseases includes those in a unit dose form which means a physically separated and formed medicament suitable for administration, and contains the polypeptides in a daily dose or in a dose from 1/40 to several folds (up to 4 folds) of the daily dose. Examples of such medicaments are injections, liquids, powders, granules, tablets, capsules, sublinguals, ophthalmic solutions, nasal drops and suppositories.

The present agent can be administered to patients orally or parenterally. In both administrations, the agent exerts a satisfactory effect in the treatment and/or the prevention for the susceptive diseases. Although it varies dependently on the types of susceptive diseases and their symptoms, the agent can be orally administered to patients or parenterally administered to patients' intradermal tissues, subcutaneous tissues, muscles, and veins at a dose as amounts of the polypeptides in the range of about 0.1 μg-500 mg/shot, preferably, about 0.1-100 mg/shot, 1-4 times/day or 1-7 times/week, for one day to one year. The present agent for susceptive diseases further includes the forms by applying gene therapy. When a transformant into which the DNAs encoding the polypeptides of this invention are introduced are administered to patients to express in them, they exert equivalent effects as above administrations. For example, "Jikken-Igaku Bessatsu, Bio-manual Up Series, Idenshi-Chiryo-No-Kisogijutsu (Basic Techniques for Gene Therapy)" (1996), edited by Takashi SHIMADA, Izumi SAITO and Takaya OZAWA, published by Yodosha, Tokyo, Japan, details the general procedures for the gene therapy.

The biological activities and acute toxicity of the present polypeptides are explained based on Experiment 3 and 4 below, respectively.

EXPERIMENT 3

Biological Activity

EXPERIMENT 3-1

Antitumor Effect in vitro

A human histocytic lymphoma cell line U937 (ATCC CRL-1593), and a cell line Molt4 (ATCC CRL-1582), derived from human T lymphoblasts, were subcultured in RPMI 1640 medium containing 10 v/v % bovine fetal serum. The cells collected by centrifugation from each subculturing system in logarithmic phase were suspended in the same medium to give a concentration of 2×10⁵ cells/ml. Every one ml of the each cell suspension was charged into each of 13 wells of multiwell plates with 24 wells, "3047", commercialized by Becton Dickinson Labware, New Jersey, U.S.A. After each of dilutions of 12 types of the purified polypeptides prepared in Example A-1 to A-4 with PBS was further charged into the each well, the cells were cultured at 37° C. for 72 hours in a 5 v/v % CO₂ incubator. The final concentration of each of the purified polypeptides was one unit/ml as an L-asparaginase activity. As a control, after charged with equivalent volume of PBS, the cells were cultured correspondingly. The cells were collected after the cultivation to stain cells died with trypan blue. Cell survival ratio in each systems with the purified polypeptides was compared with that in the control. All of the cell survival ratios with the purified polypeptides were significantly lower than that in the control. These results indicate that all of the present polypeptides, obtained in Examples A-1 to A-4, have cytotoxicity to U937 and Molt4.

EXPERIMENT 3-2

Antitumor Effect in vivo

For model mice were used C3H mice wherein a mouse lymphoma cell line 6C3HED, registered in Cell Resource Center for Biomedical Research, Institute of Development Aging and Cancer, Tohoku University, Sendai, Japan, was transplanted with passages by subcutaneous injections at their sides in a range of 1×10⁷ cells/body every 8 days in usual manner. To the model mice were administered the purified polypeptides obtained in Example A-1 to A-4 in the range of 400 unit/body by venoclyses every day from fourth to seventh days after transplanted with the cells. Dimensions of the tumors were observed with naked eyes on fourth and eighth day after the transplantations. The purified polypeptides were administered after diluted with 0.15 M NaCl and filtrated with membrane filters, 0.45 μm in pore size, commercialized by Millipore Corp., Bedford, U.S.A. As a control, 0.15 M NaCl was treated correspondingly. While significant enlargements of the tumors were observed in the control, significant involutions or disappearances of the tumors were observed in mice administered with the polypeptides. These results indicates that all of the present polypeptides, obtained in Examples A-1 to A-4, are able to cure the tumors of model mice.

EXPERIMENT 4

Acute Toxicity

The purified polypeptides obtained in Examples A-1 to A-4 were separately administered to 8-week-old mice percutaneously, perorally or intraperitoneally according to conventional manner. The LD₅₀ of all the polypeptides was about 100 mg/kg or higher independently of the administration routes. These results evidenced that the present polypeptides could be safely incorporated into pharmaceuticals for administering human.

The following examples explain the present agent for susceptive diseases.

EXAMPLE B-1

Solution

The purified polypeptides obtained in Examples A-1 to A-4 were separately dissolved to give a concentration of 0.1 mg/ml in physiological saline containing one w/v % human serum albumin as a stabilizer, and sterilized with membrane filters according to conventional manner to obtain solutions.

All of the products have satisfactory stabilities and can be used as injections, ophthalmic solutions, collunarium in the treatment and/or the prevention of susceptive diseases including a malignant tumor, acute leukemia, malignant lymphoma, an acute transformation of chronic leukemia, T-lymphocytic leukemia.

EXAMPLE B-2

Solution

The purified polypeptides obtained in Examples A-1 to A-4 were separately dissolved to give a concentration of 0.1 mg/ml in physiological saline containing one w/v % glycerol as a stabilizer, and sterilized with membrane filters according to conventional manner to obtain solutions.

All of the products have satisfactory stabilities and can be used as injections, ophthalmic solutions, collunarium for the treatment and/or the prevention of susceptive diseases including a malignant tumor, acute leukemia, malignant lymphoma, an acute transformation of chronic leukemia and T-lymphocytic leukemia.

EXAMPLE B-3

Dry Injection

The purified polypeptides obtained in Examples A-1 to A-4 were separately dissolved to give a concentration of 50 mg/ml in physiological saline containing one w/v % purified gelatin as a stabilizer, and the solutions were sterilized with membrane filters according to conventional manner. One ml aliquots of the sterilized solutions were distributed to vials, lyophilized and cap sealed.

All of the products have satisfactory stabilities and can be used as dry injections for the treatment and/or the prevention of susceptive diseases including a malignant tumor, acute leukemia, malignant lymphoma, an acute transformation of chronic leukemia and T-lymphocytic leukemia.

EXAMPLE B-4

Ointment

"HI-BIS-WAKO 104", a carboxyvinyl polymer commercialized by Wako Pure Chemicals, Tokyo, Japan, and a purified trehalose were dissolved in sterilized distilled water to give concentrations of 1.4 w/w % and 2.0 w/w %, respectively, and the purified polypeptides obtained in Examples A-1 to A-4 were separately mixed to homogeneity in the solutions followed by adjusting the pH of the resulting solutions to pH 7.2 to obtain pastes containing about one mg/g of the polypeptides.

All of the products have satisfactory spreadabilities and stabilities, and can be used as ointments for treating and/or preventing susceptive diseases including a malignant tumor, acute leukemia, malignant lymphoma, an acute transformation of chronic leukemia and T-lymphocytic leukemia.

EXAMPLE B-5

Tablet

Any one of the purified polypeptides obtained in Examples A-1 to A-4 and LUMIN, i.e. [bis-4-(1-ethylquinoline)][γ-4'-(1-ethylquinoline] pentamethionine cyanine, as a cell activator were mixed to homogeneity with "FINETOSE®", an hydrous crystalline α-maltose commercialized by Hayashibara Co.,Ltd., Okayama, Japan, and the mixtures were tabletted by tabletting machine to obtain tablets, about 200 mg weight each, containing the polypeptide and the LUMIN, about 5 mg each.

All of the products have satisfactory swallowing abilities, stabilities and cell activating activities, and can be used for treating and/or preventing susceptive diseases including a malignant tumor, acute leukemia, malignant lymphoma, an acute transformation of chronic leukemia and T-lymphocytic leukemia.

The present invention is based on the findings of polypeptides which originate from mammal, having L-asparaginase activity. The polypeptides are substances which have revealed amino acid sequences totally, and stable activities to hydrolyze L-asparagine. Therefore, the polypeptides exert satisfactory effects in the treatment and/or the prevention for diseases caused by tumor cells dependent on L-asparagine.

The polypeptides originate from mammal, so that they have low antigenicities to human and don't cause serious side effects even when administered in large amounts or continuously. Therefore, the polypeptides have the advantage that they can exert desired effects without restricted controls on patients'sensitivities.

The polypeptides thus valuable can be produced in desired amounts using the present DNAs encoding them.

Thus, the present invention is a significant invention which has a remarkable effect and gives a great contribution to this field.

While there has been described what is at present considered to be the preferred embodiments of the present invention, it will be understood the various modifications may be made therein, and it is intended to cover in the appended claims all such modifications as fall within the true spirits and scope of the invention.

    __________________________________________________________________________     #             SEQUENCE LISTING                                                    - -  - - (1) GENERAL INFORMATION:                                              - -    (iii) NUMBER OF SEQUENCES: 50                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:4 amino acid - #s                                                   (B) TYPE:amino acid                                                            (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                   - -     (ii) MOLECULE TYPE:peptide                                             - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:1:                                 - - Thr Gly Gly Thr                                                           1                                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:5 amino acid - #s                                                   (B) TYPE:amino acid                                                            (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                   - -     (ii) MOLECULE TYPE:peptide                                             - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:2:                                 - - His Gly Thr Asp Thr                                                       1               5                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:5 amino acid - #s                                                   (B) TYPE:amino acid                                                            (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                   - -     (ii) MOLECULE TYPE:peptide                                             - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:3:                                 - - Gln Cys Leu Xaa Gly                                                       1               5                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:363 amino ac - #ids                                                 (B) TYPE:amino acid                                                            (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                   - -     (ii) MOLECULE TYPE:peptide                                             - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:4:                                 - - Met Ala Arg Ala Ser Gly Ser Glu Arg His Le - #u Leu Leu Ile Tyr         Thr                                                                              1               5   - #                10  - #                15               - - Gly Gly Thr Leu Gly Met Gln Ser Lys Gly Gl - #y Val Leu Val Pro Gly                   20      - #            25      - #            30                    - - Pro Gly Leu Val Thr Leu Leu Arg Thr Leu Pr - #o Met Phe His Asp Lys               35          - #        40          - #        45                        - - Glu Phe Ala Gln Ala Gln Gly Leu Pro Asp Hi - #s Ala Leu Ala Leu Pro           50              - #    55              - #    60                            - - Pro Ala Ser His Gly Pro Arg Val Leu Tyr Th - #r Val Leu Glu Cys Gln       65                  - #70                  - #75                  - #80         - - Pro Leu Leu Asp Ser Ser Asp Met Thr Ile As - #p Asp Trp Ile Arg Ile                       85  - #                90  - #                95                - - Ala Lys Ile Ile Glu Arg His Tyr Glu Gln Ty - #r Gln Gly Phe Val Val                   100      - #           105      - #           110                   - - Ile His Gly Thr Asp Thr Met Ala Phe Gly Al - #a Ser Met Leu Ser Phe               115          - #       120          - #       125                       - - Met Leu Glu Asn Leu His Lys Pro Val Ile Le - #u Thr Gly Ala Gln Val           130              - #   135              - #   140                           - - Pro Ile Arg Val Leu Trp Asn Asp Ala Arg Gl - #u Asn Leu Leu Gly Ala       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Leu Leu Val Ala Gly Gln Tyr Ile Ile Pro Gl - #u Val Cys Leu Phe         Met                                                                                              165  - #               170  - #               175              - - Asn Ser Gln Leu Phe Arg Gly Asn Arg Val Th - #r Lys Val Asp Ser Gln                   180      - #           185      - #           190                   - - Lys Phe Glu Ala Phe Cys Ser Pro Asn Leu Se - #r Pro Leu Ala Thr Val               195          - #       200          - #       205                       - - Gly Ala Asp Val Thr Ile Ala Trp Asp Leu Va - #l Arg Lys Val Asn Trp           210              - #   215              - #   220                           - - Lys Asp Pro Leu Val Val His Ser Asn Met Gl - #u His Asp Val Ala Leu       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Leu Arg Leu Tyr Pro Gly Ile Pro Ala Ser Le - #u Val Arg Ala Phe         Leu                                                                                              245  - #               250  - #               255              - - Gln Pro Pro Leu Lys Gly Val Val Leu Glu Th - #r Phe Gly Ser Gly Asn                   260      - #           265      - #           270                   - - Gly Pro Ser Lys Pro Asp Leu Leu Gln Glu Le - #u Arg Ala Ala Ala Gln               275          - #       280          - #       285                       - - Arg Gly Leu Ile Met Val Asn Cys Ser Gln Cy - #s Leu Arg Gly Ser Val           290              - #   295              - #   300                           - - Thr Pro Gly Tyr Ala Thr Ser Leu Ala Gly Al - #a Asn Ile Val Ser Gly       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Leu Asp Met Thr Ser Glu Ala Ala Leu Ala Ly - #s Leu Ser Tyr Val         Leu                                                                                              325  - #               330  - #               335              - - Gly Leu Pro Glu Leu Ser Leu Glu Arg Arg Gl - #n Glu Leu Leu Ala Lys                   340      - #           345      - #           350                   - - Asp Leu Arg Gly Glu Met Thr Leu Pro Thr Al - #a                                   355          - #       360         363                                  - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:565 amino ac - #ids                                                 (B) TYPE:amino acid                                                            (D) TOPOLOGY:linear                                                   - -     (ii) MOLECULE TYPE:peptide                                             - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:5:                                 - - Met Ala Arg Ala Ser Gly Ser Glu Arg His Le - #u Leu Leu Ile Tyr Thr       1               5   - #                10  - #                15                - - Gly Gly Thr Leu Gly Met Gln Ser Lys Gly Gl - #y Val Leu Val Pro Gly                   20      - #            25      - #            30                    - - Pro Gly Leu Val Thr Leu Leu Arg Thr Leu Pr - #o Met Phe His Asp Lys               35          - #        40          - #        45                        - - Glu Phe Ala Gln Ala Gln Gly Leu Pro Asp Hi - #s Ala Leu Ala Leu Pro           50              - #    55              - #    60                            - - Pro Ala Ser His Gly Pro Arg Val Leu Tyr Th - #r Val Leu Glu Cys Gln       65                  - #70                  - #75                  - #80         - - Pro Leu Leu Asp Ser Ser Asp Met Thr Ile As - #p Asp Trp Ile Arg Ile                       85  - #                90  - #                95                - - Ala Lys Ile Ile Glu Arg His Tyr Glu Gln Ty - #r Gln Gly Phe Val Val                   100      - #           105      - #           110                   - - Ile His Gly Thr Asp Thr Met Ala Phe Gly Al - #a Ser Met Leu Ser Phe               115          - #       120          - #       125                       - - Met Leu Glu Asn Leu His Lys Pro Val Ile Le - #u Thr Gly Ala Gln Val           130              - #   135              - #   140                           - - Pro Ile Arg Val Leu Trp Asn Asp Ala Arg Gl - #u Asn Leu Leu Gly Ala       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Leu Leu Val Ala Gly Gln Tyr Ile Ile Pro Gl - #u Val Cys Leu Phe         Met                                                                                              165  - #               170  - #               175              - - Asn Ser Gln Leu Phe Arg Gly Asn Arg Val Th - #r Lys Val Asp Ser Gln                   180      - #           185      - #           190                   - - Lys Phe Glu Ala Phe Cys Ser Pro Asn Leu Se - #r Pro Leu Ala Thr Val               195          - #       200          - #       205                       - - Gly Ala Asp Val Thr Ile Ala Trp Asp Leu Va - #l Arg Lys Val Asn Trp           210              - #   215              - #   220                           - - Lys Asp Pro Leu Val Val His Ser Asn Met Gl - #u His Asp Val Ala Leu       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Leu Arg Leu Tyr Pro Gly Ile Pro Ala Ser Le - #u Val Arg Ala Phe         Leu                                                                                              245  - #               250  - #               255              - - Gln Pro Pro Leu Lys Gly Val Val Leu Glu Th - #r Phe Gly Ser Gly Asn                   260      - #           265      - #           270                   - - Gly Pro Ser Lys Pro Asp Leu Leu Gln Glu Le - #u Arg Ala Ala Ala Gln               275          - #       280          - #       285                       - - Arg Gly Leu Ile Met Val Asn Cys Ser Gln Cy - #s Leu Arg Gly Ser Val           290              - #   295              - #   300                           - - Thr Pro Gly Tyr Ala Thr Ser Leu Ala Gly Al - #a Asn Ile Val Ser Gly       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Leu Asp Met Thr Ser Glu Ala Ala Leu Ala Ly - #s Leu Ser Tyr Val         Leu                                                                                              325  - #               330  - #               335              - - Gly Leu Pro Glu Leu Ser Leu Glu Arg Arg Gl - #n Glu Leu Leu Ala Lys                   340      - #           345      - #           350                   - - Asp Leu Arg Gly Glu Met Thr Leu Pro Thr Al - #a Asp Leu His Gln Ser               355          - #       360          - #       365                       - - Ser Pro Pro Gly Ser Thr Leu Gly Gln Gly Va - #l Ala Arg Leu Phe Ser           370              - #   375              - #   380                           - - Leu Phe Gly Cys Gln Glu Glu Asp Ser Val Gl - #n Asp Ala Val Met Pro       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Ser Leu Ala Leu Ala Leu Ala His Ala Gly Gl - #u Leu Glu Ala Leu         Gln                                                                                              405  - #               410  - #               415              - - Ala Leu Met Glu Leu Gly Ser Asp Leu Arg Le - #u Lys Asp Ser Asn Gly                   420      - #           425      - #           430                   - - Gln Thr Leu Leu His Val Ala Ala Arg Asn Gl - #y Arg Asp Gly Val Val               435          - #       440          - #       445                       - - Thr Met Leu Leu His Arg Gly Met Asp Val As - #n Ala Arg Asp Arg Asp           450              - #   455              - #   460                           - - Gly Leu Ser Pro Leu Leu Leu Ala Val Gln Gl - #y Arg His Arg Glu Cys       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Ile Arg Leu Leu Arg Lys Ala Gly Ala Cys Le - #u Ser Pro Gln Asp         Leu                                                                                              485  - #               490  - #               495              - - Lys Asp Ala Gly Thr Glu Leu Cys Arg Leu Al - #a Ser Arg Ala Asp Met                   500      - #           505      - #           510                   - - Glu Gly Leu Gln Ala Trp Gly Gln Ala Gly Al - #a Asp Leu Gln Gln Pro               515          - #       520          - #       525                       - - Gly Tyr Asp Gly Arg Ser Ala Leu Cys Val Al - #a Glu Ala Ala Gly Asn           530              - #   535              - #   540                           - - Gln Glu Val Leu Ala Leu Leu Arg Asn Leu Al - #a Leu Val Gly Pro Glu       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Val Pro Pro Ala Ile                                                                       565                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:365 amino ac - #ids                                                 (B) TYPE:amino acid                                                            (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                   - -     (ii) MOLECULE TYPE:peptide                                             - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:6:                                 - - Met Ala Arg Ala Val Gly Pro Glu Arg Arg Le - #u Leu Ala Val Tyr         Thr                                                                              1               5   - #                10  - #                15               - - Gly Gly Thr Ile Gly Met Arg Ser Glu Leu Gl - #y Val Leu Val Pro Gly                   20      - #            25      - #            30                    - - Thr Gly Leu Ala Ala Ile Leu Arg Thr Leu Pr - #o Met Phe His Asp Glu               35          - #        40          - #        45                        - - Glu His Ala Arg Ala Arg Gly Leu Ser Glu As - #p Thr Leu Val Leu Pro           50              - #    55              - #    60                            - - Pro Asp Ser Arg Asn Gln Arg Ile Leu Tyr Th - #r Val Leu Glu Cys Gln       65                  - #70                  - #75                  - #80         - - Pro Leu Phe Asp Ser Ser Asp Met Thr Ile Al - #a Glu Trp Val Arg Val                       85  - #                90  - #               95                 - - Ala Gln Thr Ile Lys Arg His Tyr Glu Gln Ty - #r His Gly Phe Val Val                   100      - #           105      - #           110                   - - Ile His Gly Thr Asp Thr Met Ala Phe Ala Al - #a Ser Met Leu Ser Phe               115          - #       120          - #       125                       - - Met Leu Glu Asn Leu Gln Lys Thr Val Ile Le - #u Thr Gly Ala Gln Val           130              - #   135              - #   140                           - - Pro Ile His Ala Leu Trp Ser Asp Gly Arg Gl - #u Asn Leu Leu Gly Ala       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Leu Leu Met Ala Gly Gln Tyr Val Ile Pro Gl - #u Val Cys Leu Phe         Phe                                                                                              165  - #               170  - #               175              - - Gln Asn Gln Leu Phe Arg Gly Asn Arg Ala Th - #r Lys Val Asp Ala Arg                   180      - #           185      - #           190                   - - Arg Phe Ala Ala Phe Cys Ser Pro Asn Leu Le - #u Pro Leu Ala Thr Val               195          - #       200          - #       205                       - - Gly Ala Asp Ile Thr Ile Asn Arg Glu Leu Va - #l Arg Lys Val Asp Gly           210              - #   215              - #   220                           - - Lys Ala Gly Leu Val Val His Ser Ser Met Gl - #u Gln Asp Val Gly Leu       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Leu Arg Leu Tyr Pro Gly Ile Pro Ala Ala Le - #u Val Arg Ala Phe         Leu                                                                                              245  - #               250  - #               255              - - Gln Pro Pro Leu Lys Gly Val Val Met Glu Th - #r Phe Gly Ser Gly Asn                   260      - #           265      - #           270                   - - Gly Pro Thr Lys Pro Asp Leu Leu Gln Glu Le - #u Arg Val Ala Thr Glu               275          - #       280          - #       285                       - - Arg Gly Leu Val Ile Val Asn Cys Thr Gln Cy - #s Leu Arg Gly Ala Val           290              - #   295              - #   300                           - - Thr Thr Asp Tyr Ala Ala Gly Met Ala Met Al - #a Gly Ala Asn Val Ile       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Ser Gly Phe Asp Met Thr Ser Glu Ala Ala Le - #u Ala Lys Leu Ser         Tyr                                                                                              325  - #               330  - #               335              - - Val Leu Gly Gln Pro Gly Leu Ser Leu Asp Va - #l Arg Lys Glu Leu Leu                   340      - #           345      - #           350                   - - Thr Lys Asp Leu Arg Gly Glu Met Thr Pro Pr - #o Ser Val                           355          - #       360          - #       365                       - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:365 amino ac - #ids                                                 (B) TYPE:amino acid                                                            (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                   - -     (ii) MOLECULE TYPE:peptide                                             - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:7:                                 - - Met Ala Arg Ala Val Gly Pro Glu Arg Arg Le - #u Leu Ala Val Tyr Thr       1               5   - #                10  - #                15                - - Gly Gly Thr Ile Gly Met Arg Ser Glu Leu Gl - #y Val Leu Val Pro Gly                   20      - #            25      - #            30                    - - Thr Gly Leu Ala Ala Ile Leu Arg Thr Leu Pr - #o Met Phe His Asp Glu               35          - #        40          - #        45                        - - Glu His Ala Arg Ala Arg Gly Leu Ser Glu As - #p Thr Leu Val Leu Pro           50              - #    55              - #    60                            - - Pro Asp Ser Arg Asn Gln Arg Ile Leu Tyr Th - #r Val Leu Glu Cys Gln       65                  - #70                  - #75                  - #80         - - Pro Leu Phe Asp Ser Ser Asp Met Thr Ile Al - #a Glu Trp Val Arg Val                       85  - #                90  - #                95                - - Ala Gln Thr Ile Lys Arg His Tyr Glu Gln Ty - #r His Gly Phe Val Val                   100      - #           105      - #           110                   - - Ile His Gly Thr Asp Thr Met Ala Phe Ala Al - #a Ser Met Leu Ser Phe               115          - #       120          - #       125                       - - Met Leu Glu Asn Leu Gln Lys Thr Val Ile Le - #u Thr Gly Ala Gln Val           130              - #   135              - #   140                           - - Pro Ile His Ala Leu Trp Ser Asp Gly Arg Gl - #u Asn Leu Leu Gly Ala       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Leu Leu Met Ala Gly Gln Tyr Val Ile Pro Gl - #u Val Cys Leu Phe         Phe                                                                                              165  - #               170  - #               175              - - Gln Asn Gln Leu Phe Arg Gly Asn Arg Ala Th - #r Lys Val Asp Ala Arg                   180      - #           185      - #           190                   - - Arg Phe Ala Ala Phe Cys Ser Pro Asn Leu Le - #u Pro Leu Ala Thr Val               195          - #       200          - #       205                       - - Gly Ala Asp Ile Thr Ile Asn Arg Glu Leu Va - #l Arg Lys Val Asp Gly           210              - #   215              - #   220                           - - Lys Ala Gly Leu Val Val His Ser Ser Met Gl - #u Gln Asp Val Gly Leu       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Leu Arg Leu Tyr Pro Gly Ile Pro Ala Ala Le - #u Val Arg Ala Phe         Leu                                                                                              245  - #               250  - #               255              - - Gln Pro Pro Leu Lys Gly Val Val Met Glu Th - #r Phe Gly Ser Gly Asn                   260      - #           265      - #           270                   - - Gly Pro Thr Lys Pro Asp Leu Leu Gln Glu Le - #u Arg Val Ala Thr Glu               275          - #       280          - #       285                       - - Arg Gly Leu Val Ile Val Asn Cys Thr Gln Cy - #s Leu Arg Gly Ala Val           290              - #   295              - #   300                           - - Thr Thr Asp Tyr Ala Ala Gly Met Ala Met Al - #a Gly Ala Gly Val Ile       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Ser Gly Phe Asp Met Thr Ser Glu Ala Ala Le - #u Ala Lys Leu Ser         Tyr                                                                                              325  - #               330  - #               335              - - Val Leu Gly Gln Pro Gly Leu Ser Leu Asp Va - #l Arg Lys Glu Leu Leu                   340      - #           345      - #           350                   - - Thr Lys Asp Leu Arg Gly Glu Met Thr Pro Pr - #o Ser Val                           355          - #       360          - #       365                       - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:365 amino ac - #ids                                                 (B) TYPE:amino acid                                                            (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                   - -     (ii) MOLECULE TYPE:peptide                                             - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:8:                                 - - Met Ala Arg Ala Val Gly Pro Glu Arg Arg Le - #u Leu Ala Val Tyr Thr       1               5   - #                10  - #                15                - - Gly Gly Thr Ile Gly Met Arg Ser Glu Leu Gl - #y Val Leu Val Pro Gly                   20      - #            25      - #            30                    - - Thr Gly Leu Ala Ala Ile Leu Arg Thr Leu Pr - #o Met Phe His Asp Glu               35          - #        40          - #        45                        - - Glu His Ala Arg Ala Arg Gly Leu Ser Glu As - #p Thr Leu Val Leu Pro           50              - #    55              - #    60                            - - Pro Asp Ser Arg Asn Gln Arg Ile Leu Tyr Th - #r Val Leu Glu Cys Gln       65                  - #70                  - #75                  - #80         - - Pro Leu Phe Asp Ser Ser Asp Met Thr Ile Al - #a Glu Trp Val Arg Val                       85  - #                90  - #                95                - - Ala Gln Thr Ile Lys Arg His Tyr Glu Gln Ty - #r His Gly Phe Val Val                   100      - #           105      - #           110                   - - Ile His Gly Thr Asp Thr Met Ala Phe Ala Al - #a Ser Met Leu Ser Phe               115          - #       120          - #       125                       - - Met Leu Glu Asn Leu Gln Lys Thr Val Ile Le - #u Thr Gly Ala Gln Val           130              - #   135              - #   140                           - - Pro Ile His Ala Leu Trp Ser Asp Gly Arg Gl - #u Asn Leu Leu Gly Ala       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Leu Leu Met Ala Gly Gln Tyr Val Ile Pro Gl - #u Val Cys Leu Phe         Phe                                                                                              165  - #               170  - #               175              - - Gln Asn Gln Leu Phe Arg Gly Asn Arg Ala Th - #r Lys Val Asp Ala Arg                   180      - #           185      - #           190                   - - Arg Phe Ala Ala Phe Cys Ser Pro Asn Leu Le - #u Pro Leu Ala Thr Val               195          - #       200          - #       205                       - - Gly Ala Asp Ile Thr Ile Asn Arg Glu Leu Va - #l Arg Lys Val Asp Gly           210              - #   215              - #   220                           - - Lys Ala Gly Leu Val Val His Ser Ser Met Gl - #u Gln Asp Val Gly Leu       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Leu Arg Leu Tyr Pro Gly Ile Pro Ala Ala Le - #u Val Arg Ala Phe         Leu                                                                                              245  - #               250  - #               255              - - Gln Pro Pro Leu Lys Gly Val Val Met Glu Th - #r Phe Gly Ser Gly Asn                   260      - #           265      - #           270                   - - Gly Pro Thr Lys Pro Asp Leu Leu Gln Glu Le - #u Arg Val Ala Thr Glu               275          - #       280          - #       285                       - - Arg Gly Leu Val Ile Val Asn Cys Thr Gln Cy - #s Leu Gln Gly Ala Val           290              - #   295              - #   300                           - - Thr Thr Asp Tyr Ala Ala Gly Met Ala Met Al - #a Gly Ala Asn Val Ile       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Ser Gly Phe Asp Met Thr Ser Glu Ala Ala Le - #u Ala Lys Leu Ser         Tyr                                                                                              325  - #               330  - #               335              - - Val Leu Gly Gln Pro Gly Leu Ser Leu Asp Va - #l Arg Lys Glu Leu Leu                   340      - #           345      - #           350                   - - Thr Lys Asp Leu Arg Gly Glu Met Thr Pro Pr - #o Ser Val                           355          - #       360          - #       365                       - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:365 amino ac - #ids                                                 (B) TYPE:amino acid                                                            (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                   - -     (ii) MOLECULE TYPE:peptide                                             - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:9:                                 - - Met Ala Arg Ala Val Gly Pro Glu Arg Arg Le - #u Leu Ala Val Tyr Thr       1               5   - #                10  - #                15                - - Gly Gly Thr Ile Gly Met Arg Ser Glu Leu Gl - #y Val Leu Val Pro Gly                   20      - #            25      - #            30                    - - Thr Gly Leu Ala Ala Ile Leu Arg Thr Leu Pr - #o Met Phe His Asp Glu               35          - #        40          - #        45                        - - Glu His Ala Arg Ala Arg Gly Leu Ser Glu As - #p Thr Leu Val Leu Pro           50              - #    55              - #    60                            - - Pro Asp Ser Arg Asn Gln Arg Ile Leu Tyr Th - #r Val Leu Glu Cys Gln       65                  - #70                  - #75                  - #80         - - Pro Leu Phe Asp Ser Ser Asp Met Thr Ile Al - #a Glu Trp Val Arg Val                       85  - #                90  - #                95                - - Ala Gln Thr Ile Lys Arg His Tyr Glu Gln Ty - #r His Gly Phe Val Val                   100      - #           105      - #           110                   - - Ile His Gly Thr Asp Thr Met Ala Phe Ala Al - #a Ser Met Leu Ser Phe               115          - #       120          - #       125                       - - Met Leu Glu Asn Leu Gln Lys Thr Val Ile Le - #u Thr Gly Ala Gln Val           130              - #   135              - #   140                           - - Pro Ile His Ala Leu Trp Ser Asp Gly Arg Gl - #u Asn Leu Leu Gly Ala       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Leu Leu Met Ala Gly Gln Tyr Val Ile Pro Gl - #u Val Cys Leu Phe         Phe                                                                                              165  - #               170  - #               175              - - Gln Asn Gln Leu Phe Arg Gly Asn Arg Ala Th - #r Lys Val Asp Ala Arg                   180      - #           185      - #           190                   - - Arg Phe Ala Ala Phe Cys Ser Pro Asn Leu Le - #u Pro Leu Ala Thr Val               195          - #       200          - #       205                       - - Gly Ala Asp Ile Thr Ile Asn Arg Glu Leu Va - #l Arg Lys Val Asp Gly           210              - #   215              - #   220                           - - Lys Ala Gly Leu Val Val His Ser Ser Met Gl - #u Gln Asp Val Gly Leu       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Leu Arg Leu Tyr Pro Gly Ile Pro Ala Ala Le - #u Val Arg Ala Phe         Leu                                                                                              245  - #               250  - #               255              - - Gln Pro Pro Leu Lys Gly Val Val Met Glu Th - #r Phe Gly Ser Gly Asn                   260      - #           265      - #           270                   - - Gly Pro Thr Lys Pro Asp Leu Leu Gln Glu Le - #u Arg Val Ala Thr Glu               275          - #       280          - #       285                       - - Arg Gly Leu Val Ile Val Asn Cys Thr Gln Cy - #s Leu Gln Gly Ala Val           290              - #   295              - #   300                           - - Thr Thr Asp Tyr Ala Ala Gly Met Ala Met Al - #a Gly Ala Gly Val Ile       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Ser Gly Phe Asp Met Thr Ser Glu Ala Ala Le - #u Ala Lys Leu Ser         Tyr                                                                                              325  - #               330  - #               335              - - Val Leu Gly Gln Pro Gly Leu Ser Leu Asp Va - #l Arg Lys Glu Leu Leu                   340      - #           345      - #           350                   - - Thr Lys Asp Leu Arg Gly Glu Met Thr Pro Pr - #o Ser Val                           355          - #       360          - #       365                       - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:1089 base pa - #irs                                                 (B) TYPE:nucleic acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                   - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:10:                                - - ATGGCGCGCG CATCAGGCTC CGAGAGGCAC CTGCTGCTCA TCTACACTGG CG -              #GCACTTTG     60                                                                  - - GGCATGCAGA GCAAGGGCGG GGTGCTCGTC CCCGGCCCAG GCCTGGTCAC TC -             #TGCTGCGG    120                                                                  - - ACCCTGCCCA TGTTCCATGA CAAGGAGTTC GCCCAGGCCC AGGGCCTCCC TG -             #ACCATGCT    180                                                                  - - CTGGCGCTGC CCCCTGCCAG CCACGGCCCC AGGGTCCTCT ACACGGTGCT GG -             #AGTGCCAG    240                                                                  - - CCCCTCTTGG ATTCCAGCGA CATGACCATC GATGATTGGA TTCGCATAGC CA -             #AGATCATA    300                                                                  - - GAGAGGCACT ATGAGCAGTA CCAAGGCTTT GTGGTTATCC ACGGCACCGA CA -             #CCATGGCC    360                                                                  - - TTTGGGGCCT CCATGCTGTC CTTCATGCTG GAAAACCTGC ACAAACCAGT CA -             #TCCTCACT    420                                                                  - - GGCGCCCAGG TGCCAATCCG TGTGCTGTGG AATGACGCCC GGGAAAACCT GC -             #TGGGGGCG    480                                                                  - - TTGCTTGTGG CCGGCCAATA CATCATCCCT GAGGTCTGCC TGTTTATGAA CA -             #GTCAGCTG    540                                                                  - - TTTCGGGGAA ACCGGGTAAC CAAGGTGGAC TCCCAGAAGT TTGAGGCCTT CT -             #GCTCCCCC    600                                                                  - - AATCTGTCCC CACTAGCCAC TGTGGGCGCG GATGTCACAA TTGCCTGGGA CC -             #TGGTGCGC    660                                                                  - - AAGGTCAACT GGAAGGACCC GCTGGTGGTG CACAGCAACA TGGAGCACGA CG -             #TGGCACTG    720                                                                  - - CTGCGCCTCT ACCCTGGCAT CCCGGCCTCC CTGGTCCGGG CATTCCTGCA GC -             #CCCCGCTC    780                                                                  - - AAGGGCGTGG TCCTGGAGAC CTTCGGCTCT GGCAACGGGC CGAGCAAGCC CG -             #ACCTGCTG    840                                                                  - - CAGGAGTTGC GGGCCGCGGC CCAGCGCGGC CTCATCATGG TCAACTGCAG CC -             #AGTGCCTG    900                                                                  - - CGGGGGTCTG TGACCCCGGG CTATGCCACG AGCTTGGCGG GCGCCAACAT CG -             #TGTCCGGC    960                                                                  - - TTAGACATGA CCTCAGAGGC CGCGCTGGCT AAGCTGTCCT ACGTGTTGGG CC -             #TGCCGGAG   1020                                                                  - - CTGAGCCTGG AGCGCAGGCA GGAGCTGCTG GCCAAGGATC TTCGCGGGGA AA -             #TGACACTG   1080                                                                  - - CCCACGGCA                - #                  - #                       - #       1089                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:1095 base pa - #irs                                                 (B) TYPE:nucleic acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                   - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:11:                                - - ATGGCGCGCG CGGTGGGGCC CGAGCGGAGG CTGCTGGCCG TCTACACCGG CG -              #GCACCATT     60                                                                  - - GGCATGCGGA GTGAGCTCGG CGTGCTTGTG CCCGGGACGG GCCTGGCTGC CA -             #TCCTGAGG    120                                                                  - - ACACTGCCCA TGTTCCATGA CGAGGAGCAC GCCCGAGCCC GCGGCCTCTC TG -             #AGGACACC    180                                                                  - - CTGGTGCTAC CCCCGGACAG CCGCAACCAG AGGATCCTCT ACACCGTGCT GG -             #AGTGCCAG    240                                                                  - - CCCCTCTTCG ACTCCAGTGA CATGACCATC GCTGAGTGGG TTCGCGTTGC CC -             #AGACCATC    300                                                                  - - AAGAGGCACT ACGAGCAGTA CCACGGCTTT GTGGTCATCC ACGGCACCGA CA -             #CCATGGCC    360                                                                  - - TTTGCTGCCT CGATGCTGTC CTTCATGCTG GAGAACCTGC AGAAGACTGT CA -             #TCCTCACT    420                                                                  - - GGGGCCCAGG TGCCCATCCA TGCCCTGTGG AGCGACGGCC GTGAGAACCT GC -             #TGGGGGCA    480                                                                  - - CTGCTCATGG CTGGCCAGTA TGTGATCCCA GAGGTCTGCC TTTTCTTCCA GA -             #ATCAGCTG    540                                                                  - - TTTCGGGGCA ACCGGGCAAC CAAGGTAGAC GCTCGGAGGT TCGCAGCTTT CT -             #GCTCCCCG    600                                                                  - - AACCTGCTGC CTCTGGCCAC AGTGGGTGCT GACATCACAA TCAACAGGGA GC -             #TGGTGCGG    660                                                                  - - AAGGTGGACG GGAAGGCTGG GCTGGTGGTG CACAGCAGCA TGGAGCAGGA CG -             #TGGGCCTG    720                                                                  - - CTGCGCCTCT ACCCTGGGAT CCCTGCCGCC CTGGTTCGGG CCTTCTTGCA GC -             #CTCCCCTG    780                                                                  - - AAGGGCGTGG TCATGGAGAC CTTCGGTTCA GGGAACGGAC CCACCAAGCC CG -             #ACCTGCTG    840                                                                  - - CAGGAGCTGC GGGTGGCCAC CGAGCGCGGC CTGGTCATCG TCAACTGTAC CC -             #AGTGCCTC    900                                                                  - - CGGGGGGCTG TGACCACAGA CTATGCAGCT GGCATGGCCA TGGCGGGAGC CA -             #ACGTCATC    960                                                                  - - TCAGGCTTCG ACATGACATC GGAGGCCGCC CTGGCCAAGC TATCGTATGT GC -             #TGGGCCAG   1020                                                                  - - CCAGGGCTGA GCCTGGATGT CAGGAAGGAG CTGCTGACCA AGGACCTTCG GG -             #GGGAGATG   1080                                                                  - - ACGCCACCCT CGGTG              - #                  - #                       - #  1095                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:1095 base pa - #irs                                                 (B) TYPE:nucleic acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                   - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:12:                                - - ATGGCGCGCG CGGTGGGGCC CGAGCGGAGG CTGCTGGCCG TCTACACCGG CG -              #GCACCATT    60                                                                   - - GGCATGCGGA GTGAGCTCGG CGTGCTTGTG CCCGGGACGG GCCTGGCTGC CA -             #TCCTGAGG    120                                                                  - - ACACTGCCCA TGTTCCATGA CGAGGAGCAC GCCCGAGCCC GCGGCCTCTC TG -             #AGGACACC    180                                                                  - - CTGGTGCTAC CCCCGGACAG CCGCAACCAG AGGATCCTCT ACACCGTGCT GG -             #AGTGCCAG    240                                                                  - - CCCCTCTTCG ACTCCAGTGA CATGACCATC GCTGAGTGGG TTCGCGTTGC CC -             #AGACCATC    300                                                                  - - AAGAGGCACT ACGAGCAGTA CCACGGCTTT GTGGTCATCC ACGGCACCGA CA -             #CCATGGCC    360                                                                  - - TTTGCTGCCT CGATGCTGTC CTTCATGCTG GAGAACCTGC AGAAGACTGT CA -             #TCCTCACT    420                                                                  - - GGGGCCCAGG TGCCCATCCA TGCCCTGTGG AGCGACGGCC GTGAGAACCT GC -             #TGGGGGCA    480                                                                  - - CTGCTCATGG CTGGCCAGTA TGTGATCCCA GAGGTCTGCC TTTTCTTCCA GA -             #ATCAGCTG    540                                                                  - - TTTCGGGGCA ACCGGGCAAC CAAGGTAGAC GCTCGGAGGT TCGCAGCTTT CT -             #GCTCCCCG    600                                                                  - - AACCTGCTGC CTCTGGCCAC AGTGGGTGCT GACATCACAA TCAACAGGGA GC -             #TGGTGCGG    660                                                                  - - AAGGTGGACG GGAAGGCTGG GCTGGTGGTG CACAGCAGCA TGGAGCAGGA CG -             #TGGGCCTG    720                                                                  - - CTGCGCCTCT ACCCTGGGAT CCCTGCCGCC CTGGTTCGGG CCTTCTTGCA GC -             #CTCCCCTG    780                                                                  - - AAGGGCGTGG TCATGGAGAC CTTCGGTTCA GGGAACGGAC CCACCAAGCC CG -             #ACCTGCTG    840                                                                  - - CAGGAGCTGC GGGTGGCCAC CGAGCGCGGC CTGGTCATCG TCAACTGTAC CC -             #AGTGCCTC    900                                                                  - - CGGGGGGCTG TGACCACAGA CTATGCAGCT GGCATGGCCA TGGCGGGAGC CG -             #GCGTCATC    960                                                                  - - TCAGGCTTCG ACATGACATC GGAGGCCGCC CTGGCCAAGC TATCGTATGT GC -             #TGGGCCAG   1020                                                                  - - CCAGGGCTGA GCCTGGATGT CAGGAAGGAG CTGCTGACCA AGGACCTTCG GG -             #GGGAGATG   1080                                                                  - - ACGCCACCCT CGGTG              - #                  - #                       - #  1095                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:1095 base pa - #irs                                                 (B) TYPE:nucleic acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                   - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:13:                                - - ATGGCGCGCG CGGTGGGGCC CGAGCGGAGG CTGCTGGCCG TCTACACCGG CG -              #GCACCATT    60                                                                   - - GGCATGCGGA GTGAGCTCGG CGTGCTTGTG CCCGGGACGG GCCTGGCTGC CA -             #TCCTGAGG    120                                                                  - - ACACTGCCCA TGTTCCATGA CGAGGAGCAC GCCCGAGCCC GCGGCCTCTC TG -             #AGGACACC    180                                                                  - - CTGGTGCTAC CCCCGGACAG CCGCAACCAG AGGATCCTCT ACACCGTGCT GG -             #AGTGCCAG    240                                                                  - - CCCCTCTTCG ACTCCAGTGA CATGACCATC GCTGAGTGGG TTCGCGTTGC CC -             #AGACCATC    300                                                                  - - AAGAGGCACT ACGAGCAGTA CCACGGCTTT GTGGTCATCC ACGGCACCGA CA -             #CCATGGCC    360                                                                  - - TTTGCTGCCT CGATGCTGTC CTTCATGCTG GAGAACCTGC AGAAGACTGT CA -             #TCCTCACT    420                                                                  - - GGGGCCCAGG TGCCCATCCA TGCCCTGTGG AGCGACGGCC GTGAGAACCT GC -             #TGGGGGCA    480                                                                  - - CTGCTCATGG CTGGCCAGTA TGTGATCCCA GAGGTCTGCC TTTTCTTCCA GA -             #ATCAGCTG    540                                                                  - - TTTCGGGGCA ACCGGGCAAC CAAGGTAGAC GCTCGGAGGT TCGCAGCTTT CT -             #GCTCCCCG    600                                                                  - - AACCTGCTGC CTCTGGCCAC AGTGGGTGCT GACATCACAA TCAACAGGGA GC -             #TGGTGCGG    660                                                                  - - AAGGTGGACG GGAAGGCTGG GCTGGTGGTG CACAGCAGCA TGGAGCAGGA CG -             #TGGGCCTG    720                                                                  - - CTGCGCCTCT ACCCTGGGAT CCCTGCCGCC CTGGTTCGGG CCTTCTTGCA GC -             #CTCCCCTG    780                                                                  - - AAGGGCGTGG TCATGGAGAC CTTCGGTTCA GGGAACGGAC CCACCAAGCC CG -             #ACCTGCTG    840                                                                  - - CAGGAGCTGC GGGTGGCCAC CGAGCGCGGC CTGGTCATCG TCAACTGTAC CC -             #AGTGCCTC    900                                                                  - - CAGGGGGCTG TGACCACAGA CTATGCAGCT GGCATGGCCA TGGCGGGAGC CA -             #ACGTCATC    960                                                                  - - TCAGGCTTCG ACATGACATC GGAGGCCGCC CTGGCCAAGC TATCGTATGT GC -             #TGGGCCAG   1020                                                                  - - CCAGGGCTGA GCCTGGATGT CAGGAAGGAG CTGCTGACCA AGGACCTTCG GG -             #GGGAGATG   1080                                                                  - - ACGCCACCCT CGGTG              - #                  - #                       - #  1095                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:1095 base pa - #irs                                                 (B) TYPE:nucleic acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                   - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:14:                                - - ATGGCGCGCG CGGTGGGGCC CGAGCGGAGG CTGCTGGCCG TCTACACCGG CG -              #GCACCATT     60                                                                  - - GGCATGCGGA GTGAGCTCGG CGTGCTTGTG CCCGGGACGG GCCTGGCTGC CA -             #TCCTGAGG    120                                                                  - - ACACTGCCCA TGTTCCATGA CGAGGAGCAC GCCCGAGCCC GCGGCCTCTC TG -             #AGGACACC    180                                                                  - - CTGGTGCTAC CCCCGGACAG CCGCAACCAG AGGATCCTCT ACACCGTGCT GG -             #AGTGCCAG    240                                                                  - - CCCCTCTTCG ACTCCAGTGA CATGACCATC GCTGAGTGGG TTCGCGTTGC CC -             #AGACCATC    300                                                                  - - AAGAGGCACT ACGAGCAGTA CCACGGCTTT GTGGTCATCC ACGGCACCGA CA -             #CCATGGCC    360                                                                  - - TTTGCTGCCT CGATGCTGTC CTTCATGCTG GAGAACCTGC AGAAGACTGT CA -             #TCCTCACT    420                                                                  - - GGGGCCCAGG TGCCCATCCA TGCCCTGTGG AGCGACGGCC GTGAGAACCT GC -             #TGGGGGCA    480                                                                  - - CTGCTCATGG CTGGCCAGTA TGTGATCCCA GAGGTCTGCC TTTTCTTCCA GA -             #ATCAGCTG    540                                                                  - - TTTCGGGGCA ACCGGGCAAC CAAGGTAGAC GCTCGGAGGT TCGCAGCTTT CT -             #GCTCCCCG    600                                                                  - - AACCTGCTGC CTCTGGCCAC AGTGGGTGCT GACATCACAA TCAACAGGGA GC -             #TGGTGCGG    660                                                                  - - AAGGTGGACG GGAAGGCTGG GCTGGTGGTG CACAGCAGCA TGGAGCAGGA CG -             #TGGGCCTG    720                                                                  - - CTGCGCCTCT ACCCTGGGAT CCCTGCCGCC CTGGTTCGGG CCTTCTTGCA GC -             #CTCCCCTG    780                                                                  - - AAGGGCGTGG TCATGGAGAC CTTCGGTTCA GGGAACGGAC CCACCAAGCC CG -             #ACCTGCTG    840                                                                  - - CAGGAGCTGC GGGTGGCCAC CGAGCGCGGC CTGGTCATCG TCAACTGTAC CC -             #AGTGCCTC    900                                                                  - - CAGGGGGCTG TGACCACAGA CTATGCAGCT GGCATGGCCA TGGCGGGAGC CG -             #GCGTCATC    960                                                                  - - TCAGGCTTCG ACATGACATC GGAGGCCGCC CTGGCCAAGC TATCGTATGT GC -             #TGGGCCAG   1020                                                                  - - CCAGGGCTGA GCCTGGATGT CAGGAAGGAG CTGCTGACCA AGGACCTTCG GG -             #GGGAGATG   1080                                                                  - - ACGCCACCCT CGGTG              - #                  - #                       - #  1095                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:1928 base pa - #irs                                                 (B) TYPE:nucleic acid                                                          (C) STRANDEDNESS:double                                                        (D) TOPOLOGY:linear                                                   - -     (ii) MOLECULE TYPE:cDNA to mRNA                                        - -    (iii) HYPOTHETICAL:No                                                   - -     (iv) ANTI-SENSE:No                                                     - -     (vi) ORIGINAL SOURCE:                                                           (A) ORGANISM:guinea pig                                                        (F) TISSUE TYPE:liver                                                 - -     (iX) FEATURE:                                                                   (A) NAME/KEY:5'UTR                                                             (B) LOCATION:1..19                                                             (C) IDENTIFICATION METHOD: - #S                                                (A) NAME/KEY:mat peptide                                                       (B) LOCATION:20..1714                                                          (C) IDENTIFICATION METHOD: - #S                                                (A) NAME/KEY:3'UTR                                                             (B) LOCATION:1715..1928                                                        (C) IDENTIFICATION METHOD: - #S                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                               - - GAGTGGCTTA GCCGCAGGC ATG GCG CGC GCA TCA GGC TCC - # GAG AGG CAC               49                                                                                          - #   Met Ala Arg Ala Ser Gly Ser Glu Arg - # His                              - #   1               - #5                  - # 10            - - CTG CTG CTC ATC TAC ACT GGC GGC ACT TTG GG - #C ATG CAG AGC AAG GGC            97                                                                        Leu Leu Leu Ile Tyr Thr Gly Gly Thr Leu Gl - #y Met Gln Ser Lys Gly                            15  - #                20  - #                25                - - GGG GTG CTC GTC CCC GGC CCA GGC CTG GTC AC - #T CTG CTG CGG ACC CTG           145                                                                        Gly Val Leu Val Pro Gly Pro Gly Leu Val Th - #r Leu Leu Arg Thr Leu                        30      - #            35      - #            40                    - - CCC ATG TTC CAT GAC AAG GAG TTC GCC CAG GC - #C CAG GGC CTC CCT GAC           193                                                                        Pro Met Phe His Asp Lys Glu Phe Ala Gln Al - #a Gln Gly Leu Pro Asp                    45          - #        50          - #        55                        - - CAT GCT CTG GCG CTG CCC CCT GCC AGC CAC GG - #C CCC AGG GTC CTC TAC           241                                                                        His Ala Leu Ala Leu Pro Pro Ala Ser His Gl - #y Pro Arg Val Leu Tyr                60              - #    65              - #    70                            - - ACG GTG CTG GAG TGC CAG CCC CTC TTG GAT TC - #C AGC GAC ATG ACC ATC           289                                                                        Thr Val Leu Glu Cys Gln Pro Leu Leu Asp Se - #r Ser Asp Met Thr Ile            75                  - #80                  - #85                  - #90         - - GAT GAT TGG ATT CGC ATA GCC AAG ATC ATA GA - #G AGG CAC TAT GAG CAG           337                                                                        Asp Asp Trp Ile Arg Ile Ala Lys Ile Ile Gl - #u Arg His Tyr Glu Gln                            95  - #                100 - #                105               - - TAC CAA GGC TTT GTG GTT ATC CAC GGC ACC GA - #C ACC ATG GCC TTT GGG           385                                                                        Tyr Gln Gly Phe Val Val Ile His Gly Thr As - #p Thr Met Ala Phe Gly                        110      - #           115      - #           120                   - - GCC TCC ATG CTG TCC TTC ATG CTG GAA AAC CT - #G CAC AAA CCA GTC ATC           433                                                                        Ala Ser Met Leu Ser Phe Met Leu Glu Asn Le - #u His Lys Pro Val Ile                    125          - #       130          - #       135                       - - CTC ACT GGC GCC CAG GTG CCA ATC CGT GTG CT - #G TGG AAT GAC GCC CGG           481                                                                        Leu Thr Gly Ala Gln Val Pro Ile Arg Val Le - #u Trp Asn Asp Ala Arg                140              - #   145              - #   150                           - - GAA AAC CTG CTG GGG GCG TTG CTT GTG GCC GG - #C CAA TAC ATC ATC CCT           529                                                                        Glu Asn Leu Leu Gly Ala Leu Leu Val Ala Gl - #y Gln Tyr Ile Ile Pro            155                 1 - #60                 1 - #65                 1 -       #70                                                                               - - GAG GTC TGC CTG TTT ATG AAC AGT CAG CTG TT - #T CGG GGA AAC CGG         GTA      577                                                                     Glu Val Cys Leu Phe Met Asn Ser Gln Leu Ph - #e Arg Gly Asn Arg Val                           175  - #               180  - #               185               - - ACC AAG GTG GAC TCC CAG AAG TTT GAG GCC TT - #C TGC TCC CCC AAT CTG           625                                                                        Thr Lys Val Asp Ser Gln Lys Phe Glu Ala Ph - #e Cys Ser Pro Asn Leu                        190      - #           195      - #           200                   - - TCC CCA CTA GCC ACT GTG GGC GCG GAT GTC AC - #A ATT GCC TGG GAC CTG           673                                                                        Ser Pro Leu Ala Thr Val Gly Ala Asp Val Th - #r Ile Ala Trp Asp Leu                    205          - #       210          - #       215                       - - GTG CGC AAG GTC AAC TGG AAG GAC CCG CTG GT - #G GTG CAC AGC AAC ATG           721                                                                        Val Arg Lys Val Asn Trp Lys Asp Pro Leu Va - #l Val His Ser Asn Met                220              - #   225              - #   230                           - - GAG CAC GAC GTG GCA CTG CTG CGC CTC TAC CC - #T GGC ATC CCG GCC TCC           769                                                                        Glu His Asp Val Ala Leu Leu Arg Leu Tyr Pr - #o Gly Ile Pro Ala Ser            235                 2 - #40                 2 - #45                 2 -       #50                                                                               - - CTG GTC CGG GCA TTC CTG CAG CCC CCG CTC AA - #G GGC GTG GTC CTG         GAG      817                                                                     Leu Val Arg Ala Phe Leu Gln Pro Pro Leu Ly - #s Gly Val Val Leu Glu                           255  - #               260  - #               265               - - ACC TTC GGC TCT GGC AAC GGG CCG AGC AAG CC - #C GAC CTG CTG CAG GAG           865                                                                        Thr Phe Gly Ser Gly Asn Gly Pro Ser Lys Pr - #o Asp Leu Leu Gln Glu                        270      - #           275      - #           280                   - - TTG CGG GCC GCG GCC CAG CGC GGC CTC ATC AT - #G GTC AAC TGC AGC CAG           913                                                                        Leu Arg Ala Ala Ala Gln Arg Gly Leu Ile Me - #t Val Asn Cys Ser Gln                    285          - #       290          - #       295                       - - TGC CTG CGG GGG TCT GTG ACC CCG GGC TAT GC - #C ACG AGC TTG GCG GGC           961                                                                        Cys Leu Arg Gly Ser Val Thr Pro Gly Tyr Al - #a Thr Ser Leu Ala Gly                300              - #   305              - #   310                           - - GCC AAC ATC GTG TCC GGC TTA GAC ATG ACC TC - #A GAG GCC GCG CTG GCT          1009                                                                        Ala Asn Ile Val Ser Gly Leu Asp Met Thr Se - #r Glu Ala Ala Leu Ala            315                 3 - #20                 3 - #25                 3 -       #30                                                                               - - AAG CTG TCC TAC GTG TTG GGC CTG CCG GAG CT - #G AGC CTG GAG CGC         AGG     1057                                                                     Lys Leu Ser Tyr Val Leu Gly Leu Pro Glu Le - #u Ser Leu Glu Arg Arg                           335  - #               340  - #               345               - - CAG GAG CTG CTG GCC AAG GAT CTT CGC GGG GA - #A ATG ACA CTG CCC ACG          1105                                                                        Gln Glu Leu Leu Ala Lys Asp Leu Arg Gly Gl - #u Met Thr Leu Pro Thr                        350      - #           355      - #           360                   - - GCA GAC CTG CAC CAG TCC TCT CCG CCG GGC AG - #C ACA CTG GGG CAA GGT          1153                                                                        Ala Asp Leu His Gln Ser Ser Pro Pro Gly Se - #r Thr Leu Gly Gln Gly                    365          - #       370          - #       375                       - - GTC GCC CGG CTC TTT AGT CTG TTC GGT TGC CA - #G GAG GAA GAT TCG GTG          1201                                                                        Val Ala Arg Leu Phe Ser Leu Phe Gly Cys Gl - #n Glu Glu Asp Ser Val                380              - #   385              - #   390                           - - CAG GAC GCC GTG ATG CCC AGC CTG GCC CTG GC - #C TTG GCC CAT GCT GGT          1249                                                                        Gln Asp Ala Val Met Pro Ser Leu Ala Leu Al - #a Leu Ala His Ala Gly            395                 4 - #00                 4 - #05                 4 -       #10                                                                               - - GAA CTC GAG GCT CTG CAG GCA CTT ATG GAG CT - #G GGC AGT GAC CTG         CGC     1297                                                                     Glu Leu Glu Ala Leu Gln Ala Leu Met Glu Le - #u Gly Ser Asp Leu Arg                           415  - #               420  - #               425               - - CTA AAG GAC TCT AAT GGC CAA ACC CTG TTG CA - #T GTG GCT GCT CGG AAT          1345                                                                        Leu Lys Asp Ser Asn Gly Gln Thr Leu Leu Hi - #s Val Ala Ala Arg Asn                        430      - #           435      - #           440                   - - GGG CGT GAT GGC GTG GTC ACC ATG CTG CTG CA - #C AGA GGC ATG GAT GTC          1393                                                                        Gly Arg Asp Gly Val Val Thr Met Leu Leu Hi - #s Arg Gly Met Asp Val                    445          - #       450          - #       455                       - - AAT GCC CGA GAC CGA GAC GGC CTC AGC CCA CT - #G CTG TTG GCT GTA CAG          1441                                                                        Asn Ala Arg Asp Arg Asp Gly Leu Ser Pro Le - #u Leu Leu Ala Val Gln                460              - #   465              - #   470                           - - GGC AGG CAT CGG GAA TGC ATC AGG CTG CTG CG - #G AAG GCT GGG GCC TGC          1489                                                                        Gly Arg His Arg Glu Cys Ile Arg Leu Leu Ar - #g Lys Ala Gly Ala Cys            475                 4 - #80                 4 - #85                 4 -       #90                                                                               - - CTG TCC CCC CAG GAC CTG AAG GAT GCA GGG AC - #C GAG CTG TGC AGG         CTG     1537                                                                     Leu Ser Pro Gln Asp Leu Lys Asp Ala Gly Th - #r Glu Leu Cys Arg Leu                           495  - #               500  - #               505               - - GCA TCC AGG GCT GAC ATG GAA GGC CTG CAG GC - #A TGG GGG CAG GCT GGG          1585                                                                        Ala Ser Arg Ala Asp Met Glu Gly Leu Gln Al - #a Trp Gly Gln Ala Gly                        510      - #           515      - #           520                   - - GCC GAC CTG CAG CAG CCG GGC TAT GAT GGG CG - #C AGC GCT CTG TGT GTC          1633                                                                        Ala Asp Leu Gln Gln Pro Gly Tyr Asp Gly Ar - #g Ser Ala Leu Cys Val                    525          - #       530          - #       535                       - - GCA GAA GCA GCC GGG AAC CAG GAG GTG CTG GC - #C CTT CTG CGG AAC CTG          1681                                                                        Ala Glu Ala Ala Gly Asn Gln Glu Val Leu Al - #a Leu Leu Arg Asn Leu                540              - #   545              - #   550                           - - GCA CTT GTA GGC CCG GAA GTG CCG CCT GCC AT - #C TGATCGCCAG CAATCCCGC     T   1734                                                                        Ala Leu Val Gly Pro Glu Val Pro Pro Ala Il - #e                                555                 5 - #60                 5 - #65                             - - GTGGTGTGAG CCACTCCGCC ATCTGCTGCT TTGACCCACT CGAGGGACCC TA -              #GCACACGA   1794                                                                  - - CCCCCCAGCA GGATGCACCC CACTACTTAG AGTATACCCC AGGCTGGCTC AG -             #TGACAAGC   1854                                                                  - - TGCAAAGGTC TTTGTTGGCA GAACAGCAAT AAAGTAACTA CAGAGTGGCC AA -             #AAAAAAAA   1914                                                                  - - AAAAAAAAAA AAAA              - #                  - #                       - #   1928                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:2096 base pa - #irs                                                 (B) TYPE:nucleic acid                                                          (C) STRANDEDNESS:double                                                        (D) TOPOLOGY:linear                                                   - -     (ii) MOLECULE TYPE:cDNA to mRNA                                        - -    (iii) HYPOTHETICAL:No                                                   - -     (iv) ANTI-SENSE:No                                                     - -     (vi) ORIGINAL SOURCE:                                                           (A) ORGANISM:human                                                             (F) TISSUE TYPE:liver                                                 - -     (iX) FEATURE:                                                                   (A) NAME/KEY:5'UTR                                                             (B) LOCATION:1..92                                                             (C) IDENTIFICATION METHOD: - #S                                                (A) NAME/KEY:mat peptide                                                       (B) LOCATION:93..1811                                                          (C) IDENTIFICATION METHOD: - #S                                                (A) NAME/KEY:3'UTR                                                             (B) LOCATION:1812..2096                                                        (C) IDENTIFICATION METHOD: - #S                                       - -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:16:                     - - CGCCCCGGGC CTCCTCCGCG CAGTCCCTGA GTCCCGCAGG CCCTGCGTCC CC -              #GCTGCACA     60                                                                  - - CCCCCGTCCA CTCCCGTGGT CCCCGGTCCG GC ATG GCG CGC GCG - # GTG GGG         CCC      113                                                                                       - #                  - #Met Ala Arg Ala Val Gly Pro                           - #                  - #1               5                     - - GAG CGG AGG CTG CTG GCC GTC TAC ACC GGC GG - #C ACC ATT GGC ATG CGG           161                                                                        Glu Arg Arg Leu Leu Ala Val Tyr Thr Gly Gl - #y Thr Ile Gly Met Arg                    10          - #        15          - #        20                        - - AGT GAG CTC GGC GTG CTT GTG CCC GGG ACG GG - #C CTG GCT GCC ATC CTG           209                                                                        Ser Glu Leu Gly Val Leu Val Pro Gly Thr Gl - #y Leu Ala Ala Ile Leu                25              - #    30              - #    35                            - - AGG ACA CTG CCC ATG TTC CAT GAC GAG GAG CA - #C GCC CGA GCC CGC GGC           257                                                                        Arg Thr Leu Pro Met Phe His Asp Glu Glu Hi - #s Ala Arg Ala Arg Gly            40                  - #45                  - #50                  - #55         - - CTC TCT GAG GAC ACC CTG GTG CTA CCC CCG GA - #C AGC CGC AAC CAG AGG           305                                                                        Leu Ser Glu Asp Thr Leu Val Leu Pro Pro As - #p Ser Arg Asn Gln Arg             60                 - # 65                 - # 70                               - - ATC CTC TAC ACC GTG CTG GAG TGC CAG CCC CT - #C TTC GAC TCC AGT GAC           353                                                                        Ile Leu Tyr Thr Val Leu Glu Cys Gln Pro Le - #u Phe Asp Ser Ser Asp                        75      - #            80      - #            85                    - - ATG ACC ATC GCT GAG TGG GTT CGC GTT GCC CA - #G ACC ATC AAG AGG CAC           401                                                                        Met Thr Ile Ala Glu Trp Val Arg Val Ala Gl - #n Thr Ile Lys Arg His                    90          - #        95          - #        100                       - - TAC GAG CAG TAC CAC GGC TTT GTG GTC ATC CA - #C GGC ACC GAC ACC ATG           449                                                                        Tyr Glu Gln Tyr His Gly Phe Val Val Ile Hi - #s Gly Thr Asp Thr Met                105              - #   110              - #   115                           - - GCC TTT GCT GCC TCG ATG CTG TCC TTC ATG CT - #G GAG AAC CTG CAG AAG           497                                                                        Ala Phe Ala Ala Ser Met Leu Ser Phe Met Le - #u Glu Asn Leu Gln Lys            120                 1 - #25                 1 - #30                 1 -       #35                                                                               - - ACT GTC ATC CTC ACT GGG GCC CAG GTG CCC AT - #C CAT GCC CTG TGG         AGC      545                                                                     Thr Val Ile Leu Thr Gly Ala Gln Val Pro Il - #e His Ala Leu Trp Ser                           140  - #               145  - #               150               - - GAC GGC CGT GAG AAC CTG CTG GGG GCA CTG CT - #C ATG GCT GGC CAG TAT           593                                                                        Asp Gly Arg Glu Asn Leu Leu Gly Ala Leu Le - #u Met Ala Gly Gln Tyr                        155      - #           160      - #           165                   - - GTG ATC CCA GAG GTC TGC CTT TTC TTC CAG AA - #T CAG CTG TTT CGG GGC           641                                                                        Val Ile Pro Glu Val Cys Leu Phe Phe Gln As - #n Gln Leu Phe Arg Gly                    170          - #       175          - #       180                       - - AAC CGG GCA ACC AAG GTA GAC GCT CGG AGG TT - #C GCA GCT TTC TGC TCC           689                                                                        Asn Arg Ala Thr Lys Val Asp Ala Arg Arg Ph - #e Ala Ala Phe Cys Ser                185              - #   190              - #   195                           - - CCG AAC CTG CTG CCT CTG GCC ACA GTG GGT GC - #T GAC ATC ACA ATC AAC           737                                                                        Pro Asn Leu Leu Pro Leu Ala Thr Val Gly Al - #a Asp Ile Thr Ile Asn            200                 2 - #05                 2 - #10                 2 -       #15                                                                               - - AGG GAG CTG GTG CGG AAG GTG GAC GGG AAG GC - #T GGG CTG GTG GTG         CAC      785                                                                     Arg Glu Leu Val Arg Lys Val Asp Gly Lys Al - #a Gly Leu Val Val His                           220  - #               225  - #               230               - - AGC AGC ATG GAG CAG GAC GTG GGC CTG CTG CG - #C CTC TAC CCT GGG ATC           833                                                                        Ser Ser Met Glu Gln Asp Val Gly Leu Leu Ar - #g Leu Tyr Pro Gly Ile                        235      - #           240      - #           245                   - - CCT GCC GCC CTG GTT CGG GCC TTC TTG CAG CC - #T CCC CTG AAG GGC GTG           881                                                                        Pro Ala Ala Leu Val Arg Ala Phe Leu Gln Pr - #o Pro Leu Lys Gly Val                    250          - #       255          - #       260                       - - GTC ATG GAG ACC TTC GGT TCA GGG AAC GGA CC - #C ACC AAG CCC GAC CTG           929                                                                        Val Met Glu Thr Phe Gly Ser Gly Asn Gly Pr - #o Thr Lys Pro Asp Leu                265              - #   270              - #   275                           - - CTG CAG GAG CTG CGG GTG GCC ACC GAG CGC GG - #C CTG GTC ATC GTC AAC           977                                                                        Leu Gln Glu Leu Arg Val Ala Thr Glu Arg Gl - #y Leu Val Ile Val Asn            280                 2 - #85                 2 - #90                 2 -       #95                                                                               - - TGT ACC CAC TGC CTC CAG GGG GCT GTG ACC AC - #A GAC TAT GCA GCT         GGC     1025                                                                     Cys Thr His Cys Leu Gln Gly Ala Val Thr Th - #r Asp Tyr Ala Ala Gly                           300  - #               305  - #               310               - - ATG GCC ATG GCG GGA GCC GGC GTC ATC TCA GG - #C TTC GAC ATG ACA TCG          1073                                                                        Met Ala Met Ala Gly Ala Gly Val Ile Ser Gl - #y Phe Asp Met Thr Ser                        315      - #           320      - #           325                   - - GAG GCC GCC CTG GCC AAG CTA TCG TAT GTG CT - #G GGC CAG CCA GGG CTG          1121                                                                        Glu Ala Ala Leu Ala Lys Leu Ser Tyr Val Le - #u Gly Gln Pro Gly Leu                    330          - #       335          - #       340                       - - AGC CTG GAT GTC AGG AAG GAG CTG CTG ACC AA - #G GAC CTT CGG GGG GAG          1169                                                                        Ser Leu Asp Val Arg Lys Glu Leu Leu Thr Ly - #s Asp Leu Arg Gly Glu                345              - #   350              - #   355                           - - ATG ACG CCA CCC TCG GTG GAA GAG CGC CGG CC - #C TCA CTG CAG GGC AAC          1217                                                                        Met Thr Pro Pro Ser Val Glu Glu Arg Arg Pr - #o Ser Leu Gln Gly Asn            360                 3 - #65                 3 - #70                 3 -       #75                                                                               - - ACG CTG GGC GGT GGG GTC TCC TGG CTC CTC AG - #T CTG AGC GGC AGC         CAG     1265                                                                     Thr Leu Gly Gly Gly Val Ser Trp Leu Leu Se - #r Leu Ser Gly Ser Gln                           380  - #               385  - #               390               - - GAG GCA GAT GCC CTG CGG AAT GCC CTG GTG CC - #C AGC CTG GCC TGT GCT          1313                                                                        Glu Ala Asp Ala Leu Arg Asn Ala Leu Val Pr - #o Ser Leu Ala Cys Ala                        395      - #           400      - #           405                   - - GCT GCC CAC GCC GGT GAC GTG GAG GCG CTG CA - #G GCG CTT GTG GAG CTG          1361                                                                        Ala Ala His Ala Gly Asp Val Glu Ala Leu Gl - #n Ala Leu Val Glu Leu                    410          - #       415          - #       420                       - - GGC AGT GAC CTG GGC CTG GTG GAC TTT AAC GG - #C CAA ACC CCA CTG CAC          1409                                                                        Gly Ser Asp Leu Gly Leu Val Asp Phe Asn Gl - #y Gln Thr Pro Leu His                425              - #   430              - #   435                           - - GCG GCC GCC CGG GGA GGC CAC ACA GAG GCA GT - #C ACC ATG CTG CTG CAG          1457                                                                        Ala Ala Ala Arg Gly Gly His Thr Glu Ala Va - #l Thr Met Leu Leu Gln            440                 4 - #45                 4 - #50                 4 -       #55                                                                               - - AGA GGT GTG GAC GTG AAC ACC CGG GAC ACG GA - #T GGC TTC AGC CCG         CTG     1505                                                                     Arg Gly Val Asp Val Asn Thr Arg Asp Thr As - #p Gly Phe Ser Pro Leu                           460  - #               465  - #               470               - - CTG CTG GCC GTG CGG GGC AGG CAT CCG GGT GT - #C ATT GGG TTG CTG CGG          1553                                                                        Leu Leu Ala Val Arg Gly Arg His Pro Gly Va - #l Ile Gly Leu Leu Arg                        475      - #           480      - #           485                   - - GAA GCC GGG GCC TCC CTG TCC ACC CAG GAG CT - #G GAG GAA GCA GGG ACG          1601                                                                        Glu Ala Gly Ala Ser Leu Ser Thr Gln Glu Le - #u Glu Glu Ala Gly Thr                    490          - #       495          - #       500                       - - GAG CTG TGC AGG CTG GCA TAC AGG GCC GAC CT - #C GAA GGC CTG CAG GTG          1649                                                                        Glu Leu Cys Arg Leu Ala Tyr Arg Ala Asp Le - #u Glu Gly Leu Gln Val                505              - #   510              - #   515                           - - TGG TGG CAG GCA GGG GCT GAC CTG GGG CAG CC - #G GGC TAT GAC GGG CAC          1697                                                                        Trp Trp Gln Ala Gly Ala Asp Leu Gly Gln Pr - #o Gly Tyr Asp Gly His            520                 5 - #25                 5 - #30                 5 -       #35                                                                               - - AGC GCC CTG CAC GTC GCA GAG GCA GCC GGG AA - #C CTG GCA GTG GTG         GCC     1745                                                                     Ser Ala Leu His Val Ala Glu Ala Ala Gly As - #n Leu Ala Val Val Ala                           540  - #               545  - #               550               - - TTT CTA CAG AGC CTG GAG GGT GCG GTT GGT GC - #C CAG GCC CCA TGC CCA          1793                                                                        Phe Leu Gln Ser Leu Glu Gly Ala Val Gly Al - #a Gln Ala Pro Cys Pro                        555      - #           560      - #           565                   - - GAA GTG CTG CCT GGT GTC TAACCTGAAG GCGTCCTGCT GC - #AGTATAAG                 1841                                                                        Glu Val Leu Pro Gly Val                                                                570                                                                     - - CCATTCCTTC CTCCCATGAC CTGCTGGAGG GGTCTCAGGC ATGACCCCAC TG -              #CTGGGGCT   1901                                                                  - - GCTTCCCAGC CTGCTCTCAT GTAAAGCCTG AAGGCCTTTG TTGGGCAGGA CG -             #GCAATAAA   1961                                                                  - - GTCTCTGACA TCCCCTCACC AGGTCTGTAC AGCCTGGCTC TGAGAGGCTC TG -             #TCTGGGTC   2021                                                                  - - CGGGACTGTG AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA -             #AAAAAAAA   2081                                                                  - - AAAAAAAAAA AAAAA              - #                  - #                       - #  2096                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:1695 base pa - #irs                                                 (B) TYPE:nucleic acid                                                          (C) STRANDEDNESS:double                                                        (D) TOPOLOGY:linear                                                   - -     (ii) MOLECULE TYPE:cDNA to mRNA                                        - -    (iii) HYPOTHETICAL:No                                                   - -     (iv) ANTI-SENSE:No                                                     - -     (vi) ORIGINAL SOURCE:                                                           (A) ORGANISM:guinea pig                                                        (F) TISSUE TYPE:liver                                                 - -     (iX) FEATURE:                                                                   (A) NAME/KEY:mat peptide                                                       (B) LOCATION:1..1695                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                               - - ATG GCG CGC GCA TCA GGC TCC GAG AGG CAC CT - #G CTG CTC ATC TAC ACT            48                                                                        Met Ala Arg Ala Ser Gly Ser Glu Arg His Le - #u Leu Leu Ile Tyr Thr            1               5   - #                10  - #                15                - - GGC GGC ACT TTG GGC ATG CAG AGC AAG GGC GG - #G GTG CTC GTC CCC GGC            96                                                                        Gly Gly Thr Leu Gly Met Gln Ser Lys Gly Gl - #y Val Leu Val Pro Gly                        20      - #            25      - #            30                    - - CCA GGC CTG GTC ACT CTG CTG CGG ACC CTG CC - #C ATG TTC CAT GAC AAG           144                                                                        Pro Gly Leu Val Thr Leu Leu Arg Thr Leu Pr - #o Met Phe His Asp Lys                    35          - #        40          - #        45                        - - GAG TTC GCC CAG GCC CAG GGC CTC CCT GAC CA - #T GCT CTG GCG CTG CCC           192                                                                        Glu Phe Ala Gln Ala Gln Gly Leu Pro Asp Hi - #s Ala Leu Ala Leu Pro                50              - #    55              - #    60                            - - CCT GCC AGC CAC GGC CCC AGG GTC CTC TAC AC - #G GTG CTG GAG TGC CAG           240                                                                        Pro Ala Ser His Gly Pro Arg Val Leu Tyr Th - #r Val Leu Glu Cys Gln            65                  - #70                  - #75                  - #80         - - CCC CTC TTG GAT TCC AGC GAC ATG ACC ATC GA - #T GAT TGG ATT CGC ATA           288                                                                        Pro Leu Leu Asp Ser Ser Asp Met Thr Ile As - #p Asp Trp Ile Arg Ile                            85  - #                90  - #                95                - - GCC AAG ATC ATA GAG AGG CAC TAT GAG CAG TA - #C CAA GGC TTT GTG GTT           336                                                                        Ala Lys Ile Ile Glu Arg His Tyr Glu Gln Ty - #r Gln Gly Phe Val Val                        100      - #           105      - #           110                   - - ATC CAC GGC ACC GAC ACC ATG GCC TTT GGG GC - #C TCC ATG CTG TCC TTC           384                                                                        Ile His Gly Thr Asp Thr Met Ala Phe Gly Al - #a Ser Met Leu Ser Phe                    115          - #       120          - #       125                       - - ATG CTG GAA AAC CTG CAC AAA CCA GTC ATC CT - #C ACT GGC GCC CAG GTG           432                                                                        Met Leu Glu Asn Leu His Lys Pro Val Ile Le - #u Thr Gly Ala Gln Val                130              - #   135              - #   140                           - - CCA ATC CGT GTG CTG TGG AAT GAC GCC CGG GA - #A AAC CTG CTG GGG GCG           480                                                                        Pro Ile Arg Val Leu Trp Asn Asp Ala Arg Gl - #u Asn Leu Leu Gly Ala            145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - TTG CTT GTG GCC GGC CAA TAC ATC ATC CCT GA - #G GTC TGC CTG TTT         ATG      528                                                                     Leu Leu Val Ala Gly Gln Tyr Ile Ile Pro Gl - #u Val Cys Leu Phe Met                           165  - #               170  - #               175               - - AAC AGT CAG CTG TTT CGG GGA AAC CGG GTA AC - #C AAG GTG GAC TCC CAG           576                                                                        Asn Ser Gln Leu Phe Arg Gly Asn Arg Val Th - #r Lys Val Asp Ser Gln                        180      - #           185      - #           190                   - - AAG TTT GAG GCC TTC TGC TCC CCC AAT CTG TC - #C CCA CTA GCC ACT GTG           624                                                                        Lys Phe Glu Ala Phe Cys Ser Pro Asn Leu Se - #r Pro Leu Ala Thr Val                    195          - #       200          - #       205                       - - GGC GCG GAT GTC ACA ATT GCC TGG GAC CTG GT - #G CGC AAG GTC AAC TGG           672                                                                        Gly Ala Asp Val Thr Ile Ala Trp Asp Leu Va - #l Arg Lys Val Asn Trp                210              - #   215              - #   220                           - - AAG GAC CCG CTG GTG GTG CAC AGC AAC ATG GA - #G CAC GAC GTG GCA CTG           720                                                                        Lys Asp Pro Leu Val Val His Ser Asn Met Gl - #u His Asp Val Ala Leu            225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - CTG CGC CTC TAC CCT GGC ATC CCG GCC TCC CT - #G GTC CGG GCA TTC         CTG      768                                                                     Leu Arg Leu Tyr Pro Gly Ile Pro Ala Ser Le - #u Val Arg Ala Phe Leu                           245  - #               250  - #               255               - - CAG CCC CCG CTC AAG GGC GTG GTC CTG GAG AC - #C TTC GGC TCT GGC AAC           816                                                                        Gln Pro Pro Leu Lys Gly Val Val Leu Glu Th - #r Phe Gly Ser Gly Asn                        260      - #           265      - #           270                   - - GGG CCG AGC AAG CCC GAC CTG CTG CAG GAG TT - #G CGG GCC GCG GCC CAG           864                                                                        Gly Pro Ser Lys Pro Asp Leu Leu Gln Glu Le - #u Arg Ala Ala Ala Gln                    275          - #       280          - #       285                       - - CGC GGC CTC ATC ATG GTC AAC TGC AGC CAG TG - #C CTG CGG GGG TCT GTG           912                                                                        Arg Gly Leu Ile Met Val Asn Cys Ser Gln Cy - #s Leu Arg Gly Ser Val                290              - #   295              - #   300                           - - ACC CCG GGC TAT GCC ACG AGC TTG GCG GGC GC - #C AAC ATC GTG TCC GGC           960                                                                        Thr Pro Gly Tyr Ala Thr Ser Leu Ala Gly Al - #a Asn Ile Val Ser Gly            305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - TTA GAC ATG ACC TCA GAG GCC GCG CTG GCT AA - #G CTG TCC TAC GTG         TTG     1008                                                                     Leu Asp Met Thr Ser Glu Ala Ala Leu Ala Ly - #s Leu Ser Tyr Val Leu                           325  - #               330  - #               335               - - GGC CTG CCG GAG CTG AGC CTG GAG CGC AGG CA - #G GAG CTG CTG GCC AAG          1056                                                                        Gly Leu Pro Glu Leu Ser Leu Glu Arg Arg Gl - #n Glu Leu Leu Ala Lys                        340      - #           345      - #           350                   - - GAT CTT CGC GGG GAA ATG ACA CTG CCC ACG GC - #A GAC CTG CAC CAG TCC          1104                                                                        Asp Leu Arg Gly Glu Met Thr Leu Pro Thr Al - #a Asp Leu His Gln Ser                    355          - #       360          - #       365                       - - TCT CCG CCG GGC AGC ACA CTG GGG CAA GGT GT - #C GCC CGG CTC TTT AGT          1152                                                                        Ser Pro Pro Gly Ser Thr Leu Gly Gln Gly Va - #l Ala Arg Leu Phe Ser                370              - #   375              - #   380                           - - CTG TTC GGT TGC CAG GAG GAA GAT TCG GTG CA - #G GAC GCC GTG ATG CCC          1200                                                                        Leu Phe Gly Cys Gln Glu Glu Asp Ser Val Gl - #n Asp Ala Val Met Pro            385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - AGC CTG GCC CTG GCC TTG GCC CAT GCT GGT GA - #A CTC GAG GCT CTG         CAG     1248                                                                     Ser Leu Ala Leu Ala Leu Ala His Ala Gly Gl - #u Leu Glu Ala Leu Gln                           405  - #               410  - #               415               - - GCA CTT ATG GAG CTG GGC AGT GAC CTG CGC CT - #A AAG GAC TCT AAT GGC          1296                                                                        Ala Leu Met Glu Leu Gly Ser Asp Leu Arg Le - #u Lys Asp Ser Asn Gly                        420      - #           425      - #           430                   - - CAA ACC CTG TTG CAT GTG GCT GCT CGG AAT GG - #G CGT GAT GGC GTG GTC          1344                                                                        Gln Thr Leu Leu His Val Ala Ala Arg Asn Gl - #y Arg Asp Gly Val Val                    435          - #       440          - #       445                       - - ACC ATG CTG CTG CAC AGA GGC ATG GAT GTC AA - #T GCC CGA GAC CGA GAC          1392                                                                        Thr Met Leu Leu His Arg Gly Met Asp Val As - #n Ala Arg Asp Arg Asp                450              - #   455              - #   460                           - - GGC CTC AGC CCA CTG CTG TTG GCT GTA CAG GG - #C AGG CAT CGG GAA TGC          1440                                                                        Gly Leu Ser Pro Leu Leu Leu Ala Val Gln Gl - #y Arg His Arg Glu Cys            465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - ATC AGG CTG CTG CGG AAG GCT GGG GCC TGC CT - #G TCC CCC CAG GAC         CTG     1488                                                                     Ile Arg Leu Leu Arg Lys Ala Gly Ala Cys Le - #u Ser Pro Gln Asp Leu                           485  - #               490  - #               495               - - AAG GAT GCA GGG ACC GAG CTG TGC AGG CTG GC - #A TCC AGG GCT GAC ATG          1536                                                                        Lys Asp Ala Gly Thr Glu Leu Cys Arg Leu Al - #a Ser Arg Ala Asp Met                        500      - #           505      - #           510                   - - GAA GGC CTG CAG GCA TGG GGG CAG GCT GGG GC - #C GAC CTG CAG CAG CCG          1584                                                                        Glu Gly Leu Gln Ala Trp Gly Gln Ala Gly Al - #a Asp Leu Gln Gln Pro                    515          - #       520          - #       525                       - - GGC TAT GAT GGG CGC AGC GCT CTG TGT GTC GC - #A GAA GCA GCC GGG AAC          1632                                                                        Gly Tyr Asp Gly Arg Ser Ala Leu Cys Val Al - #a Glu Ala Ala Gly Asn                530              - #   535              - #   540                           - - CAG GAG GTG CTG GCC CTT CTG CGG AAC CTG GC - #A CTT GTA GGC CCG GAA          1680                                                                        Gln Glu Val Leu Ala Leu Leu Arg Asn Leu Al - #a Leu Val Gly Pro Glu            545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - GTG CCG CCT GCC ATC           - #                  - #                       - #  1695                                                                  Val Pro Pro Ala Ile                                                                            565                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:1719 base pa - #irs                                                 (B) TYPE:nucleic acid                                                          (C) STRANDEDNESS:double                                                        (D) TOPOLOGY:linear                                                   - -     (ii) MOLECULE TYPE:cDNA to mRNA                                        - -    (iii) HYPOTHETICAL:No                                                   - -     (iv) ANTI-SENSE:No                                                     - -     (vi) ORIGINAL SOURCE:                                                           (A) ORGANISM:human                                                             (F) TISSUE TYPE:liver                                                 - -     (iX) FEATURE:                                                                   (A) NAME/KEY:mat peptide                                                       (B) LOCATION:1..1719                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                               - - ATG GCG CGC GCG GTG GGG CCC GAG CGG AGG CT - #G CTG GCC GTC TAC ACC            48                                                                        Met Ala Arg Ala Val Gly Pro Glu Arg Arg Le - #u Leu Ala Val Tyr Thr            1               5   - #                10  - #                15                - - GGC GGC ACC ATT GGC ATG CGG AGT GAG CTC GG - #C GTG CTT GTG CCC GGG            96                                                                        Gly Gly Thr Ile Gly Met Arg Ser Glu Leu Gl - #y Val Leu Val Pro Gly                        20      - #            25      - #            30                    - - ACG GGC CTG GCT GCC ATC CTG AGG ACA CTG CC - #C ATG TTC CAT GAC GAG           144                                                                        Thr Gly Leu Ala Ala Ile Leu Arg Thr Leu Pr - #o Met Phe His Asp Glu                    35          - #        40          - #        45                        - - GAG CAC GCC CGA GCC CGC GGC CTC TCT GAG GA - #C ACC CTG GTG CTA CCC           192                                                                        Glu His Ala Arg Ala Arg Gly Leu Ser Glu As - #p Thr Leu Val Leu Pro                50              - #    55              - #    60                            - - CCG GAC AGC CGC AAC CAG AGG ATC CTC TAC AC - #C GTG CTG GAG TGC CAG           240                                                                        Pro Asp Ser Arg Asn Gln Arg Ile Leu Tyr Th - #r Val Leu Glu Cys Gln            65                  - #70                  - #75                  - #80         - - CCC CTC TTC GAC TCC AGT GAC ATG ACC ATC GC - #T GAG TGG GTT CGC GTT           288                                                                        Pro Leu Phe Asp Ser Ser Asp Met Thr Ile Al - #a Glu Trp Val Arg Val                            85  - #                90  - #                95                - - GCC CAG ACC ATC AAG AGG CAC TAC GAG CAG TA - #C CAC GGC TTT GTG GTC           336                                                                        Ala Gln Thr Ile Lys Arg His Tyr Glu Gln Ty - #r His Gly Phe Val Val                        100      - #           105      - #           110                   - - ATC CAC GGC ACC GAC ACC ATG GCC TTT GCT GC - #C TCG ATG CTG TCC TTC           384                                                                        Ile His Gly Thr Asp Thr Met Ala Phe Ala Al - #a Ser Met Leu Ser Phe                    115          - #       120          - #       125                       - - ATG CTG GAG AAC CTG CAG AAG ACT GTC ATC CT - #C ACT GGG GCC CAG GTG           432                                                                        Met Leu Glu Asn Leu Gln Lys Thr Val Ile Le - #u Thr Gly Ala Gln Val                130              - #   135              - #   140                           - - CCC ATC CAT GCC CTG TGG AGC GAC GGC CGT GA - #G AAC CTG CTG GGG GCA           480                                                                        Pro Ile His Ala Leu Trp Ser Asp Gly Arg Gl - #u Asn Leu Leu Gly Ala            145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - CTG CTC ATG GCT GGC CAG TAT GTG ATC CCA GA - #G GTC TGC CTT TTC         TTC      528                                                                     Leu Leu Met Ala Gly Gln Tyr Val Ile Pro Gl - #u Val Cys Leu Phe Phe                           165  - #               170  - #               175               - - CAG AAT CAG CTG TTT CGG GGC AAC CGG GCA AC - #C AAG GTA GAC GCT CGG           576                                                                        Gln Asn Gln Leu Phe Arg Gly Asn Arg Ala Th - #r Lys Val Asp Ala Arg                        180      - #           185      - #           190                   - - AGG TTC GCA GCT TTC TGC TCC CCG AAC CTG CT - #G CCT CTG GCC ACA GTG           624                                                                        Arg Phe Ala Ala Phe Cys Ser Pro Asn Leu Le - #u Pro Leu Ala Thr Val                    195          - #       200          - #       205                       - - GGT GCT GAC ATC ACA ATC AAC AGG GAG CTG GT - #G CGG AAG GTG GAC GGG           672                                                                        Gly Ala Asp Ile Thr Ile Asn Arg Glu Leu Va - #l Arg Lys Val Asp Gly                210              - #   215              - #   220                           - - AAG GCT GGG CTG GTG GTG CAC AGC AGC ATG GA - #G CAG GAC GTG GGC CTG           720                                                                        Lys Ala Gly Leu Val Val His Ser Ser Met Gl - #u Gln Asp Val Gly Leu            225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - CTG CGC CTC TAC CCT GGG ATC CCT GCC GCC CT - #G GTT CGG GCC TTC         TTG      768                                                                     Leu Arg Leu Tyr Pro Gly Ile Pro Ala Ala Le - #u Val Arg Ala Phe Leu                           245  - #               250  - #               255               - - CAG CCT CCC CTG AAG GGC GTG GTC ATG GAG AC - #C TTC GGT TCA GGG AAC           816                                                                        Gln Pro Pro Leu Lys Gly Val Val Met Glu Th - #r Phe Gly Ser Gly Asn                        260      - #           265      - #           270                   - - GGA CCC ACC AAG CCC GAC CTG CTG CAG GAG CT - #G CGG GTG GCC ACC GAG           864                                                                        Gly Pro Thr Lys Pro Asp Leu Leu Gln Glu Le - #u Arg Val Ala Thr Glu                    275          - #       280          - #       285                       - - CGC GGC CTG GTC ATC GTC AAC TGT ACC CAC TG - #C CTC CAG GGG GCT GTG           912                                                                        Arg Gly Leu Val Ile Val Asn Cys Thr His Cy - #s Leu Gln Gly Ala Val                290              - #   295              - #   300                           - - ACC ACA GAC TAT GCA GCT GGC ATG GCC ATG GC - #G GGA GCC GGC GTC ATC           960                                                                        Thr Thr Asp Tyr Ala Ala Gly Met Ala Met Al - #a Gly Ala Gly Val Ile            305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - TCA GGC TTC GAC ATG ACA TCG GAG GCC GCC CT - #G GCC AAG CTA TCG         TAT     1008                                                                     Ser Gly Phe Asp Met Thr Ser Glu Ala Ala Le - #u Ala Lys Leu Ser Tyr                           325  - #               330  - #               335               - - GTG CTG GGC CAG CCA GGG CTG AGC CTG GAT GT - #C AGG AAG GAG CTG CTG          1056                                                                        Val Leu Gly Gln Pro Gly Leu Ser Leu Asp Va - #l Arg Lys Glu Leu Leu                        340      - #           345      - #           350                   - - ACC AAG GAC CTT CGG GGG GAG ATG ACG CCA CC - #C TCG GTG GAA GAG CGC          1104                                                                        Thr Lys Asp Leu Arg Gly Glu Met Thr Pro Pr - #o Ser Val Glu Glu Arg                    355          - #       360          - #       365                       - - CGG CCC TCA CTG CAG GGC AAC ACG CTG GGC GG - #T GGG GTC TCC TGG CTC          1152                                                                        Arg Pro Ser Leu Gln Gly Asn Thr Leu Gly Gl - #y Gly Val Ser Trp Leu                370              - #   375              - #   380                           - - CTC AGT CTG AGC GGC AGC CAG GAG GCA GAT GC - #C CTG CGG AAT GCC CTG          1200                                                                        Leu Ser Leu Ser Gly Ser Gln Glu Ala Asp Al - #a Leu Arg Asn Ala Leu            385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - GTG CCC AGC CTG GCC TGT GCT GCT GCC CAC GC - #C GGT GAC GTG GAG         GCG     1248                                                                     Val Pro Ser Leu Ala Cys Ala Ala Ala His Al - #a Gly Asp Val Glu Ala                           405  - #               410  - #               415               - - CTG CAG GCG CTT GTG GAG CTG GGC AGT GAC CT - #G GGC CTG GTG GAC TTT          1296                                                                        Leu Gln Ala Leu Val Glu Leu Gly Ser Asp Le - #u Gly Leu Val Asp Phe                        420      - #           425      - #           430                   - - AAC GGC CAA ACC CCA CTG CAC GCG GCC GCC CG - #G GGA GGC CAC ACA GAG          1344                                                                        Asn Gly Gln Thr Pro Leu His Ala Ala Ala Ar - #g Gly Gly His Thr Glu                    435          - #       440          - #       445                       - - GCA GTC ACC ATG CTG CTG CAG AGA GGT GTG GA - #C GTG AAC ACC CGG GAC          1392                                                                        Ala Val Thr Met Leu Leu Gln Arg Gly Val As - #p Val Asn Thr Arg Asp                450              - #   455              - #   460                           - - ACG GAT GGC TTC AGC CCG CTG CTG CTG GCC GT - #G CGG GGC AGG CAT CCG          1440                                                                        Thr Asp Gly Phe Ser Pro Leu Leu Leu Ala Va - #l Arg Gly Arg His Pro            465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - GGT GTC ATT GGG TTG CTG CGG GAA GCC GGG GC - #C TCC CTG TCC ACC         CAG     1488                                                                     Gly Val Ile Gly Leu Leu Arg Glu Ala Gly Al - #a Ser Leu Ser Thr Gln                           485  - #               490  - #               495               - - GAG CTG GAG GAA GCA GGG ACG GAG CTG TGC AG - #G CTG GCA TAC AGG GCC          1536                                                                        Glu Leu Glu Glu Ala Gly Thr Glu Leu Cys Ar - #g Leu Ala Tyr Arg Ala                        500      - #           505      - #           510                   - - GAC CTC GAA GGC CTG CAG GTG TGG TGG CAG GC - #A GGG GCT GAC CTG GGG          1584                                                                        Asp Leu Glu Gly Leu Gln Val Trp Trp Gln Al - #a Gly Ala Asp Leu Gly                    515          - #       520          - #       525                       - - CAG CCG GGC TAT GAC GGG CAC AGC GCC CTG CA - #C GTC GCA GAG GCA GCC          1632                                                                        Gln Pro Gly Tyr Asp Gly His Ser Ala Leu Hi - #s Val Ala Glu Ala Ala                530              - #   535              - #   540                           - - GGG AAC CTG GCA GTG GTG GCC TTT CTA CAG AG - #C CTG GAG GGT GCG GTT          1680                                                                        Gly Asn Leu Ala Val Val Ala Phe Leu Gln Se - #r Leu Glu Gly Ala Val            545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - GGT GCC CAG GCC CCA TGC CCA GAA GTG CTG CC - #T GGT GTC                   - #   1719                                                                    Gly Ala Gln Ala Pro Cys Pro Glu Val Leu Pr - #o Gly Val                                        565  - #               570  - #       573                       - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 29 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                               - - AATCTCGAGC CACCATGGCG CGCGCATCA         - #                  - #                 29                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                               - - CTGCGGCCGC TTATCAGATG GCAGGCGGCA C        - #                  - #               31                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 amino - #acids                                                  (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                               - - Gly Ser Gly Asn Gly Pro Thr Lys Pro Asp Le - #u Leu Gln Glu Leu Arg       1               5   - #                10  - #                15                - - Cys                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 29 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                               - - AATCTCGAGC CACCATGGCG CGCGCGGTG         - #                  - #                 29                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                               - - CTGCGGCCGC TTATCAGACA CCAGGCAGCA C        - #                  - #               31                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                               - - CTGCGGCCGC TTATCATGCC GTGGGCAGTG T        - #                  - #               31                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                               - - CTGCGGCCGC TTATCAGCCC AACACGTAGG A        - #                  - #               31                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                               - - CTGCGGCCGC TCATTACACC GAGGGTGGCG T        - #                  - #               31                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                               - - CCCCCGGAGG CACTGGGT             - #                  - #                       - #  18                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                               - - ACCCAGTGCC TCCGGGGG             - #                  - #                       - #  18                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                               - - CCCCTGGAGG CACTGGGT             - #                  - #                       - #  18                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                               - - ACCCAGTGCC TCCAGGGG             - #                  - #                       - #  18                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                               - - CCCCCGGAGG CAGTGGGT             - #                  - #                       - #  18                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                               - - ACCCACTGCC TCCGGGGG             - #                  - #                       - #  18                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                               - - GACGTTGGCT CCCGCCAT             - #                  - #                       - #  18                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                               - - ATGGCGGGAG CCAACGTC             - #                  - #                       - #  18                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 23 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                               - - GCGAATTCAT GGCGCGCGCA TCA           - #                  - #                     23                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 26 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                               - - GCAAGCTTTC AGATGGCAGG CGGCAC          - #                  - #                   26                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 33 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                               - - GTGAATTCGG AGGTTCAGAT GGCGCGCGCA TCA       - #                  - #              33                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 28 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                               - - CTGCGGCCGC TCAGATGGCA GGCGGCAC         - #                  - #                  28                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                               - - TCGAGCCACC ATGAAGTGTT CGTGGGTTAT T        - #                  - #               31                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 30 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                               - - TTCTTCCTGA TGGCCGTAGT GACAGGAGTG         - #                  - #                30                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 30 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                               - - AATTCACTCC TGTCACTACG GCCATCAGGA         - #                  - #                30                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 31 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                               - - AGAAAATAAC CCACGAACAC TTCATGGTGG C        - #                  - #               31                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 26 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                               - - GCAAGCTTTC ATGCCGTGGG CAGTGT          - #                  - #                   26                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 23 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                               - - GCGAATTCAT GGCGCGCGCG GTG           - #                  - #                     23                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 26 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                               - - GCAAGCTTTC ACACCGAGGG TGGCGT          - #                  - #                   26                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:46:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 27 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                               - - CTGCGGCCGC TCATGCCGTG GGCAGTG          - #                  - #                  27                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:47:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                               - - CTGAATTCGG AGGTTCAGAT GGCGCGCGCG GGTG       - #                  -       #        34                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:48:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 27 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                               - - CTGCGGCCGC TCACACCGAG GGTGGCG          - #                  - #                  27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:49:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:565 amino ac - #ids                                                 (B) TYPE:amino acid                                                            (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                   - -     (ii) MOLECULE TYPE:peptide                                             - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:49:                                - - Met Ala Arg Ala Ser Gly Ser Glu Arg His Le - #u Leu Leu Ile Tyr Thr       1               5   - #                10  - #                15                - - Gly Gly Thr Leu Gly Met Gln Ser Lys Gly Gl - #y Val Leu Val Pro Gly                   20      - #            25      - #            30                    - - Pro Gly Leu Val Thr Leu Leu Arg Thr Leu Pr - #o Met Phe His Asp Lys               35          - #        40          - #        45                        - - Glu Phe Ala Gln Ala Gln Gly Leu Pro Asp Hi - #s Ala Leu Ala Leu Pro           50              - #    55              - #    60                            - - Pro Ala Ser His Gly Pro Arg Val Leu Tyr Th - #r Val Leu Glu Cys Gln       65                  - #70                  - #75                  - #80         - - Pro Leu Leu Asp Ser Ser Asp Met Thr Ile As - #p Asp Trp Ile Arg Ile                       85  - #                90  - #                95                - - Ala Lys Ile Ile Glu Arg His Tyr Glu Gln Ty - #r Gln Gly Phe Val Val                   100      - #           105      - #           110                   - - Ile His Gly Thr Asp Thr Met Ala Phe Gly Al - #a Ser Met Leu Ser Phe               115          - #       120          - #       125                       - - Met Leu Glu Asn Leu His Lys Pro Val Ile Le - #u Thr Gly Ala Gln Val           130              - #   135              - #   140                           - - Pro Ile Arg Val Leu Trp Asn Asp Ala Arg Gl - #u Asn Leu Leu Gly Ala       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Leu Leu Val Ala Gly Gln Tyr Ile Ile Pro Gl - #u Val Cys Leu Phe         Met                                                                                              165  - #               170  - #               175              - - Asn Ser Gln Leu Phe Arg Gly Asn Arg Val Th - #r Lys Val Asp Ser Gln                   180      - #           185      - #           190                   - - Lys Phe Glu Ala Phe Cys Ser Pro Asn Leu Se - #r Pro Leu Ala Thr Val               195          - #       200          - #       205                       - - Gly Ala Asp Val Thr Ile Ala Trp Asp Leu Va - #l Arg Lys Val Asn Trp           210              - #   215              - #   220                           - - Lys Asp Pro Leu Val Val His Ser Asn Met Gl - #u His Asp Val Ala Leu       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Leu Arg Leu Tyr Pro Gly Ile Pro Ala Ser Le - #u Val Arg Ala Phe         Leu                                                                                              245  - #               250  - #               255              - - Gln Pro Pro Leu Lys Gly Val Val Leu Glu Th - #r Phe Gly Ser Gly Asn                   260      - #           265      - #           270                   - - Gly Pro Ser Lys Pro Asp Leu Leu Gln Glu Le - #u Arg Ala Ala Ala Gln               275          - #       280          - #       285                       - - Arg Gly Leu Ile Met Val Asn Cys Ser Gln Cy - #s Leu Arg Gly Ser Val           290              - #   295              - #   300                           - - Thr Pro Gly Tyr Ala Thr Ser Leu Ala Gly Al - #a Asn Ile Val Ser Gly       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Leu Asp Met Thr Ser Glu Ala Ala Leu Ala Ly - #s Leu Ser Tyr Val         Leu                                                                                              325  - #               330  - #               335              - - Gly Leu Pro Glu Leu Ser Leu Glu Arg Arg Gl - #n Glu Leu Leu Ala Lys                   340      - #           345      - #           350                   - - Asp Leu Arg Gly Glu Met Thr Leu Pro Thr Al - #a Asp Leu His Gln Ser               355          - #       360          - #       365                       - - Ser Pro Pro Gly Ser Thr Leu Gly Gln Gly Va - #l Ala Arg Leu Phe Ser           370              - #   375              - #   380                           - - Leu Phe Gly Cys Gln Glu Glu Asp Ser Val Gl - #n Asp Ala Val Met Pro       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Ser Leu Ala Leu Ala Leu Ala His Ala Gly Gl - #u Leu Glu Ala Leu         Gln                                                                                              405  - #               410  - #               415              - - Ala Leu Met Glu Leu Gly Ser Asp Leu Arg Le - #u Lys Asp Ser Asn Gly                   420      - #           425      - #           430                   - - Gln Thr Leu Leu His Val Ala Ala Arg Asn Gl - #y Arg Asp Gly Val Val               435          - #       440          - #       445                       - - Thr Met Leu Leu His Arg Gly Met Asp Val As - #n Ala Arg Asp Arg Asp           450              - #   455              - #   460                           - - Gly Leu Ser Pro Leu Leu Leu Ala Val Gln Gl - #y Arg His Arg Glu Cys       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Ile Arg Leu Leu Arg Lys Ala Gly Ala Cys Le - #u Ser Pro Gln Asp         Leu                                                                                              485  - #               490  - #               495              - - Lys Asp Ala Gly Thr Glu Leu Cys Arg Leu Al - #a Ser Arg Ala Asp Met                   500      - #           505      - #           510                   - - Glu Gly Leu Gln Ala Trp Gly Gln Ala Gly Al - #a Asp Leu Gln Gln Pro               515          - #       520          - #       525                       - - Gly Tyr Asp Gly Arg Ser Ala Leu Cys Val Al - #a Glu Ala Ala Gly Asn           530              - #   535              - #   540                           - - Gln Glu Val Leu Ala Leu Leu Arg Asn Leu Al - #a Leu Val Gly Pro Glu       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Val Pro Pro Ala Ile                                                                       565                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:50:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:573 amino ac - #ids                                                 (B) TYPE:amino acid                                                            (C) STRANDEDNESS: single                                                       (D) TOPOLOGY:linear                                                   - -     (ii) MOLECULE TYPE:peptide                                             - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:50:                                - - Met Ala Arg Ala Val Gly Pro Glu Arg Arg Le - #u Leu Ala Val Tyr         Thr                                                                              1               5   - #                10  - #                15               - - Gly Gly Thr Ile Gly Met Arg Ser Glu Leu Gl - #y Val Leu Val Pro Gly                   20      - #            25      - #            30                    - - Thr Gly Leu Ala Ala Ile Leu Arg Thr Leu Pr - #o Met Phe His Asp Glu               35          - #        40          - #        45                        - - Glu His Ala Arg Ala Arg Gly Leu Ser Glu As - #p Thr Leu Val Leu Pro           50              - #    55              - #    60                            - - Pro Asp Ser Arg Asn Gln Arg Ile Leu Tyr Th - #r Val Leu Glu Cys Gln       65                  - #70                  - #75                  - #80         - - Pro Leu Phe Asp Ser Ser Asp Met Thr Ile Al - #a Glu Trp Val Arg Val                       85  - #                90  - #                95                - - Ala Gln Thr Ile Lys Arg His Tyr Glu Gln Ty - #r His Gly Phe Val Val                   100      - #           105      - #           110                   - - Ile His Gly Thr Asp Thr Met Ala Phe Ala Al - #a Ser Met Leu Ser Phe               115          - #       120          - #       125                       - - Met Leu Glu Asn Leu Gln Lys Thr Val Ile Le - #u Thr Gly Ala Gln Val           130              - #   135              - #   140                           - - Pro Ile His Ala Leu Trp Ser Asp Gly Arg Gl - #u Asn Leu Leu Gly Ala       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Leu Leu Met Ala Gly Gln Tyr Val Ile Pro Gl - #u Val Cys Leu Phe         Phe                                                                                              165  - #               170  - #               175              - - Gln Asn Gln Leu Phe Arg Gly Asn Arg Ala Th - #r Lys Val Asp Ala Arg                   180      - #           185      - #           190                   - - Arg Phe Ala Ala Phe Cys Ser Pro Asn Leu Le - #u Pro Leu Ala Thr Val               195          - #       200          - #       205                       - - Gly Ala Asp Ile Thr Ile Asn Arg Glu Leu Va - #l Arg Lys Val Asp Gly           210              - #   215              - #   220                           - - Lys Ala Gly Leu Val Val His Ser Ser Met Gl - #u Gln Asp Val Gly Leu       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Leu Arg Leu Tyr Pro Gly Ile Pro Ala Ala Le - #u Val Arg Ala Phe         Leu                                                                                              245  - #               250  - #               255              - - Gln Pro Pro Leu Lys Gly Val Val Met Glu Th - #r Phe Gly Ser Gly Asn                   260      - #           265      - #           270                   - - Gly Pro Thr Lys Pro Asp Leu Leu Gln Glu Le - #u Arg Val Ala Thr Glu               275          - #       280          - #       285                       - - Arg Gly Leu Val Ile Val Asn Cys Thr His Cy - #s Leu Gln Gly Ala Val           290              - #   295              - #   300                           - - Thr Thr Asp Tyr Ala Ala Gly Met Ala Met Al - #a Gly Ala Gly Val Ile       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Ser Gly Phe Asp Met Thr Ser Glu Ala Ala Le - #u Ala Lys Leu Ser         Tyr                                                                                              325  - #               330  - #               335              - - Val Leu Gly Gln Pro Gly Leu Ser Leu Asp Va - #l Arg Lys Glu Leu Leu                   340      - #           345      - #           350                   - - Thr Lys Asp Leu Arg Gly Glu Met Thr Pro Pr - #o Ser Val Glu Glu Arg               355          - #       360          - #       365                       - - Arg Pro Ser Leu Gln Gly Asn Thr Leu Gly Gl - #y Gly Val Ser Trp Leu           370              - #   375              - #   380                           - - Leu Ser Leu Ser Gly Ser Gln Glu Ala Asp Al - #a Leu Arg Asn Ala Leu       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Val Pro Ser Leu Ala Cys Ala Ala Ala His Al - #a Gly Asp Val Glu         Ala                                                                                              405  - #               410  - #               415              - - Leu Gln Ala Leu Val Glu Leu Gly Ser Asp Le - #u Gly Leu Val Asp Phe                   420      - #           425      - #           430                   - - Asn Gly Gln Thr Pro Leu His Ala Ala Ala Ar - #g Gly Gly His Thr Glu               435          - #       440          - #       445                       - - Ala Val Thr Met Leu Leu Gln Arg Gly Val As - #p Val Asn Thr Arg Asp           450              - #   455              - #   460                           - - Thr Asp Gly Phe Ser Pro Leu Leu Leu Ala Va - #l Arg Gly Arg His Pro       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - Gly Val Ile Gly Leu Leu Arg Glu Ala Gly Al - #a Ser Leu Ser Thr         Gln                                                                                              485  - #               490  - #               495              - - Glu Leu Glu Glu Ala Gly Thr Glu Leu Cys Ar - #g Leu Ala Tyr Arg Ala                   500      - #           505      - #           510                   - - Asp Leu Glu Gly Leu Gln Val Trp Trp Gln Al - #a Gly Ala Asp Leu Gly               515          - #       520          - #       525                       - - Gln Pro Gly Tyr Asp Gly His Ser Ala Leu Hi - #s Val Ala Glu Ala Ala           530              - #   535              - #   540                           - - Gly Asn Leu Ala Val Val Ala Phe Leu Gln Se - #r Leu Glu Gly Ala Val       545                 5 - #50                 5 - #55                 5 -       #60                                                                               - - Gly Ala Gln Ala Pro Cys Pro Glu Val Leu Pr - #o Gly Val                                   565  - #               570  - #       573                    __________________________________________________________________________ 

What is claimed is:
 1. An isolated DNA molecule encoding a polypeptide exhibiting L-asparaginase activity, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11 to 14 and fragments of any one of SEO ID NOs:11 to 14 which encode a polypeptide exhibiting L-asparaginase activity.
 2. A self-replicable vector, comprising an isolated DNA molecule encoding the polypeptide exhibiting L-asparaginase activity according to claim
 1. 3. The self-replicable vector according to claim 2, which is a plasmid vector.
 4. The self-replicable vector according to claim 2, further comprising one or more promoters selected from the group consisting of metallothionein and Tac promoters.
 5. A host cell transformed with a DNA molecule according to claim
 1. 6. A host cell according to claim 5, wherein said host cell is a member selected from the group consisting of prokaryotic and eukarvotic cells.
 7. The host cell according to claim 5, wherein said host cell is Escherichia coli.
 8. The host cell according to claim 5, wherein said host cell is a mouse cell.
 9. A host cell transformed by the self-replicable vector according to claim
 2. 10. The host cell according to claim 9, wherein said host cell is a member selected from the group consisting of prokaryotic and eukaryotic cells.
 11. The host cell according to claim 9, where in said host cell is Escherichia coli.
 12. The host cell according to claim 9, wherein said host cell is a mouse cell.
 13. A process for preparing a polypeptide exhibiting L-asparaginase activity, comprising the steps of:expressing a DNA molecule encoding the polypeptide according to claim 1; and collecting the expressed polypeptide.
 14. The process according to claim 13, wherein the expressing step comprises culturing a host cell transformed with the DNA molecule encoding the polypeptide.
 15. The process according to claim 13, wherein the collecting step comprises one or more techniques selected from the group consisting of salting out, dialysis, filtration, concentration, gel filtration chromatography, ion-exchange chomatography, affinity chromatography, hydrophobic chromatograph, isoelectric focusing and gel electrophoresis. 